Treatment of alpha-L-iduronidase (IDUA) related diseases by inhibition of natural antisense transcript to IDUA

ABSTRACT

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of Alpha-L-Iduronidase (IDUA), in particular, by targeting natural antisense polynucleotides of Alpha-L-Iduronidase (IDUA). The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of IDUA.

The present application claims the priority of U.S. Provisional PatentApplication No. 61/405,758 filed Oct. 22, 2010, which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

Embodiments of the invention comprise oligonucleotides modulatingexpression and/or function of IDUA and associated molecules.

BACKGROUND

DNA-RNA and RNA-RNA hybridization are important to many aspects ofnucleic acid function including DNA replication, transcription, andtranslation. Hybridization is also central to a variety of technologiesthat either detect a particular nucleic acid or alter its expression.Antisense nucleotides, for example, disrupt gene expression byhybridizing to target RNA, thereby interfering with RNA splicing,transcription, translation, and replication. Antisense DNA has the addedfeature that DNA-RNA hybrids serve as a substrate for digestion byribonuclease H, an activity that is present in most cell types.Antisense molecules can be delivered into cells, as is the case foroligodeoxynucleotides (ODNs), or they can be expressed from endogenousgenes as RNA molecules. The FDA recently approved an antisense drug,VITRAVENE™ (for treatment of cytomegalovirus retinitis), reflecting thatantisense has therapeutic utility.

SUMMARY

This Summary is provided to present a summary of the invention tobriefly indicate the nature and substance of the invention. It issubmitted with the understanding that it will not be used to interpretor limit the scope or meaning of the claims.

In one embodiment, the invention provides methods for inhibiting theaction of a natural antisense transcript by using antisenseoligonucleotide(s) targeted to any region of the natural antisensetranscript resulting in up-regulation of the corresponding sense gene.It is also contemplated herein that inhibition of the natural antisensetranscript can be achieved by siRNA, ribozymes and small molecules,which are considered to be within the scope of the present invention.

One embodiment provides a method of modulating function and/orexpression of an IDUA polynucleotide in biological systems, including,but not limited to, patient cells or tissues in vivo or in vitrocomprising contacting said biological system or said cells or tissueswith an antisense oligonucleotide of about 5 to about 30 nucleotides inlength wherein said oligonucleotide has at least 50% sequence identityto a reverse complement of a polynucleotide comprising 5 to 30consecutive nucleotides within nucleotides 1 to 2695 of SEQ ID NO: 2 or1 to 2082 of SEQ ID NO: 3 or 1 to 322 of SEQ ID NO: 4 or 1 to 677 of SEQID NO: 5 or 1 to 716 of SEQ ID NO: 6 or 1 to 466 of SEQ ID NO: 7 or 1 to1255 of SEQ ID NO: 8 or 1 to 2739 of SEQ ID NO: 9 thereby modulatingfunction and/or expression of the IDUA polynucleotide in said biologicalsystem including said patient cells or tissues in vivo or in vitro.

In an embodiment, an oligonucleotide targets a natural antisensesequence of IDUA polynucleotides present in a biological system, forexample, nucleotides set forth in SEQ ID NOS: 2 to 9, and any variants,alleles, homologs, mutants, derivatives, fragments and complementarysequences thereto. Examples of antisense oligonucleotides are set forthas SEQ ID NOS: 10 to 28.

Another embodiment provides a method of modulating function and/orexpression of an IDUA polynucleotide in patient cells or tissues in vivoor in vitro comprising contacting said cells or tissues with anantisense oligonucleotide 5 to 30 nucleotides in length wherein saidoligonucleotide has at least 50% sequence identity to a reversecomplement of an antisense of the IDUA polynucleotide; therebymodulating function and/or expression of the IDUA polynucleotide inpatient cells or tissues in vivo or in vitro.

Another embodiment provides a method of modulating function and/orexpression of an IDUA polynucleotide in patient cells or tissues in vivoor in vitro comprising contacting said cells or tissues with anantisense oligonucleotide 5 to 30 nucleotides in length wherein saidoligonucleotide has at least 50% sequence identity to an antisenseoligonucleotide to an IDUA polynucleotide; thereby modulating functionand/or expression of the IDUA polynucleotide in patient cells or tissuesin vivo or in vitro.

In another embodiment, the invention comprises a method of modulatingthe function or expression of an IDUA polynucleotide in a biologicalsystem comprising contacting said biological system with at least oneantisense oligonucleotide that targets a natural antisense transcript ofthe IDUA polynucleotide thereby modulating the function and/orexpression of the IDUA polynucleotide in said biological system.

In another embodiment, the invention comprises a method of modulatingthe function or expression of an IDUA polynucleotide in a biologicalsystem comprising contacting said biological system with at least oneantisense oligonucleotide that targets a region of a natural antisensetranscript of the IDUA polynucleotide thereby modulating the functionand/or expression of the IDUA polynucleotide in said biological system.

In an embodiment, the invention comprises a method of increasing thefunction and/or expression of an IDUA polynucleotide having SEQ ID NO. 1in a biological system comprising contacting said biological system withat least one antisense oligonucleotide that targets a natural antisensetranscript of said IDUA polynucleotide thereby increasing the functionand/or expression of said IDUA polynucleotide or expression productthereof.

In another embodiment, the invention comprises a method of increasingthe function and/or expression of an IDUA polynucleotide having SEQ IDNO. 1 in a biological system comprising contacting said biologicalsystem with at least one antisense oligonucleotide that targets anatural antisense transcript of said IDUA polynucleotide therebyincreasing the function and/or expression of said IDUA polynucleotide orexpression product thereof wherein the natural antisense transcripts areselected from SEQ ID NOS. 2 to 9.

In another embodiment, the invention comprises a method of method ofincreasing the function and/or expression of an IDUA polynucleotidehaving SEQ ID NO. 1 in a biological system comprising contacting saidbiological system with at least one antisense oligonucleotide thattargets a natural antisense transcript of said IDUA polynucleotidethereby increasing the function and/or expression of said IDUApolynucleotide or expression product thereof wherein the naturalantisense transcripts are selected from SEQ ID NOS. 2 to 9 and whereinthe antisense oligonucleotides are selected from at least one of SEQ IDNOS. 10 to 28.

In an embodiment, a composition comprises one or more antisenseoligonucleotides which bind to sense and/or antisense IDUApolynucleotides.

In an embodiment, the oligonucleotides comprise one or more modified orsubstituted nucleotides.

In an embodiment, the oligonucleotides comprise one or more modifiedbonds.

In yet another embodiment, the modified nucleotides comprise modifiedbases comprising phosphorothioate, methylphosphonate, peptide nucleicacids, 2′-O-methyl, fluoro- or carbon, methylene or other locked nucleicacid (LNA) molecules. Preferably, the modified nucleotides are lockednucleic acid molecules, including α-L-LNA.

In an embodiment, the oligonucleotides are administered to a patient byany delivery route including, but not limited to, orally, transdermally,via inhalation means, subcutaneously, intramuscularly, intravenously orintraperitoneally.

In an embodiment, the oligonucleotides are administered in apharmaceutical composition. A treatment regimen comprises administeringthe antisense compounds at least once to patient; however, thistreatment can be modified to include multiple doses over a period oftime. The treatment can be combined with one or more other types oftherapies.

In an embodiment, the oligonucleotides are encapsulated in a liposome orattached to a carrier molecule (e.g. cholesterol, TAT peptide).

Other aspects are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of real time PCR results showing the foldchange+standard deviation in IDUA mRNA after treatment of HepG2 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine™2000, as compared to control. Bars denoted as CUR-1820 to CUR-1823correspond to samples treated with SEQ ID NOS: 10 to 13 respectively.

FIG. 2 is a graph of real time PCR results showing the foldchange+standard deviation in IDUA mRNA after treatment of HepG2 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine™2000, as compared to control. Bars denoted as CUR-1973, CUR-1975,CUR-1976, CUR-1978, CUR-1981, CUR-1984, CUR-1985, CUR-1987, CUR-1988correspond to samples treated with SEQ ID NOS: 14 to 22 respectively.

FIG. 3 is a graph of real time PCR results showing the foldchange+standard deviation in IDUA mRNA after treatment of HcpG2 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine™2000, as compared to control. Bars denoted as CUR-1974, CUR-1977,CUR-1986, CUR-1983, CUR-1979 and CUR-1982 correspond to samples treatedwith SEQ ID NOS: 23 to 28 respectively.

FIG. 4 is a graph of real time PCR results showing the foldchange+standard deviation in human IDUA mRNA after treatment of SK-N-Ascells with phosphorothioate oligonucleotides introduced usingLipofectamine™ 2000, as compared to control. Bars denoted as CUR-1973,CUR-1975, CUR-1976, CUR-1978, CUR-1981, CUR-1984, CUR-1985, CUR-1987,CUR-1988 correspond to samples treated with SEQ ID NOS 14 to 22respectively.

FIG. 5 (SEQ ID NO: 8) shows the extension by 578 nucleotides (gray) ofthe original sequence dog DN876121 sequence (SEQ ID NO: 5) (in clear)using Clone open biosystems: NAE04B03.

Sequence Listing Description—SEQ ID NO: 1: Homo sapiens iduronidase,alpha-L-(IDUA), mRNA (NCBI Accession No.: NM_(—)000203); SEQ ID NO: 2:Natural IDUA antisense sequence (HS.656285); SEQ ID NO: 3: Natural IDUAantisense sequence (CR626108); SEQ ID NO: 4: Natural IDUA antisensesequence (DN334757); SEQ ID NO: 5: Natural IDUA antisense sequence(DN876121); SEQ ID NO: 6: Natural IDUA antisense sequence (DN744190);SEQ ID NO: 7: Natural IDUA antisense sequence (DN330918); SEQ ID NO: 8:Natural IDUA antisense sequence (DN876121-extended); SEQ ID NO: 9: humanIDUA natural antisense-extended; SEQ ID NOs: 10 to 28: Antisenseoligonucleotides. * indicates phosphothioate bond; SEQ ID NOs: 29 to 45:UniGene Cluster Hs.656285.

DETAILED DESCRIPTION

Several aspects of the invention are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. The present invention is not limited by the ordering ofacts or events, as some acts may occur in different orders and/orconcurrently with other acts or events. Furthermore, not all illustratedacts or events are required to implement a methodology in accordancewith the present invention.

All genes, gene names, and gene products disclosed herein are intendedto correspond to homologs from any species for which the compositionsand methods disclosed herein are applicable. Thus, the terms include,but are not limited to genes and gene products from humans and mice. Itis understood that when a gene or gene product from a particular speciesis disclosed, this disclosure is intended to be exemplary only, and isnot to be interpreted as a limitation unless the context in which itappears clearly indicates. Thus, for example, for the genes disclosedherein, which in some embodiments relate to mammalian nucleic acid andamino acid sequences are intended to encompass homologous and/ororthologous genes and gene products from other animals including, butnot limited to other mammals, fish, amphibians, reptiles, and birds. Inan embodiment, the genes or nucleic acid sequences are human.

DEFINITIONS

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

As used herein, the term “mRNA” means the presently known mRNAtranscript(s) of a targeted gene, and any further transcripts which maybe elucidated.

By “antisense oligonucleotides” or “antisense compound” is meant an RNAor DNA molecule that binds to another RNA or DNA (target RNA, DNA). Forexample, if it is an RNA oligonucleotide it binds to another RNA targetby means of RNA-RNA interactions and alters the activity of the targetRNA. An antisense oligonucleotide can upregulate or downregulateexpression and/or function of a particular polynucleotide. Thedefinition is meant to include any foreign RNA or DNA molecule which isuseful from a therapeutic, diagnostic, or other viewpoint. Suchmolecules include, for example, antisense RNA or DNA molecules,interference RNA (RNAi), micro RNA, decoy RNA molecules, siRNA,enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA,antisense oligomeric compounds, antisense oligonucleotides, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds that hybridize to at least aportion of the target nucleic acid. As such, these compounds may beintroduced in the form of single-stranded, double-stranded, partiallysingle-stranded, or circular oligomeric compounds.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. The term “oligonucleotide”, alsoincludes linear or circular oligomers of natural and/or modifiedmonomers or linkages, including deoxyribonucleosides, ribonucleosides,substituted and alpha-anomeric forms thereof, peptide nucleic acids(PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate,and the like. Oligonucleotides are capable of specifically binding to atarget polynucleotide by way of a regular pattern of monomer-to-monomerinteractions, such as Watson-Crick type of base pairing, Hoögsteen orreverse Hoögsteen types of base pairing, or the like.

The oligonucleotide may be “chimeric”, that is, composed of differentregions. In the context of this invention “chimeric” compounds areoligonucleotides, which contain two or more chemical regions, forexample, DNA region(s), RNA region(s), PNA region(s) etc. Each chemicalregion is made up of at least one monomer unit, i.e., a nucleotide inthe case of an oligonucleotides compound. These oligonucleotidestypically comprise at least one region wherein the oligonucleotide ismodified in order to exhibit one or more desired properties. The desiredproperties of the oligonucleotide include, but are not limited, forexample, to increased resistance to nuclease degradation, increasedcellular uptake, and/or increased binding affinity for the targetnucleic acid. Different regions of the oligonucleotide may thereforehave different properties. The chimeric oligonucleotides of the presentinvention can be formed as mixed structures of two or moreoligonucleotides, modified oligonucleotides, oligonucleosides and/oroligonucleotide analogs as described above.

The oligonucleotide can be composed of regions that can be linked in“register”, that is, when the monomers are linked consecutively, as innative DNA, or linked via spacers. The spacers are intended toconstitute a covalent “bridge” between the regions and have in preferredcases a length not exceeding about 100 carbon atoms. The spacers maycarry different functionalities, for example, having positive ornegative charge, carry special nucleic acid binding properties(intercalators, groove binders, toxins, fluorophors etc.), beinglipophilic, inducing special secondary structures like, for example,alanine containing peptides that induce alpha-helices.

As used herein “IDUA” and “Alpha-L-Iduronidase” are inclusive of allfamily members, mutants, alleles, fragments, species, coding andnoncoding sequences, sense and antisense polynucleotide strands, etc.

As used herein, the words ‘Alpha-L-iduronidase’, IDA, IDUA and MPS1, areconsidered the same in the literature and are used interchangeably inthe present application.

As used herein, the term “oligonucleotide specific for” or“oligonucleotide which targets” refers to an oligonucleotide having asequence (i) capable of forming a stable complex with a portion of thetargeted gene, or (ii) capable of forming a stable duplex with a portionof a mRNA transcript of the targeted gene. Stability of the complexesand duplexes can be determined by theoretical calculations and/or invitro assays. Exemplary assays for determining stability ofhybridization complexes and duplexes are described in the Examplesbelow.

As used herein, the term “target nucleic acid” encompasses DNA, RNA(comprising premRNA and mRNA) transcribed from such DNA, and also cDNAderived from such RNA, coding, noncoding sequences, sense or antisensepolynucleotides. The specific hybridization of an oligomeric compoundwith its target nucleic acid interferes with the normal function of thenucleic acid. This modulation of function of a target nucleic acid bycompounds, which specifically hybridize to it, is generally referred toas “antisense”. The functions of DNA to be interfered include, forexample, replication and transcription. The functions of RNA to beinterfered, include all vital functions such as, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in or facilitatedby the RNA. The overall effect of such interference with target nucleicacid function is modulation of the expression of an encoded product oroligonucleotides.

RNA interference “RNAi” is mediated by double stranded RNA (dsRNA)molecules that have sequence-specific homology to their “target” nucleicacid sequences. In certain embodiments of the present invention, themediators are 5-25 nucleotide “small interfering” RNA duplexes (siRNAs).The siRNAs are derived from the processing of dsRNA by an RNase enzymeknown as Dicer. siRNA duplex products are recruited into a multi-proteinsiRNA complex termed RISC (RNA Induced Silencing Complex). Withoutwishing to be bound by any particular theory, a RISC is then believed tobe guided to a target nucleic acid (suitably mRNA), where the siRNAduplex interacts in a sequence-specific way to mediate cleavage in acatalytic fashion. Small interfering RNAs that can be used in accordancewith the present invention can be synthesized and used according toprocedures that are well known in the art and that will be familiar tothe ordinarily skilled artisan. Small interfering RNAs for use in themethods of the present invention suitably comprise between about 1 toabout 50 nucleotides (nt). In examples of non limiting embodiments,siRNAs can comprise about 5 to about 40 nt, about 5 to about 30 nt,about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25nucleotides.

Selection of appropriate oligonucleotides is facilitated by usingcomputer programs that automatically align nucleic acid sequences andindicate regions of identity or homology. Such programs are used tocompare nucleic acid sequences obtained, for example, by searchingdatabases such as GenBank or by sequencing PCR products. Comparison ofnucleic acid sequences from a range of species allows the selection ofnucleic acid sequences that display an appropriate degree of identitybetween species. In the case of genes that have not been sequenced,Southern blots are performed to allow a determination of the degree ofidentity between genes in target species and other species. Byperforming Southern blots at varying degrees of stringency, as is wellknown in the art, it is possible to obtain an approximate measure ofidentity. These procedures allow the selection of oligonucleotides thatexhibit a high degree of complementarity to target nucleic acidsequences in a subject to be controlled and a lower degree ofcomplementarity to corresponding nucleic acid sequences in otherspecies. One skilled in the art will realize that there is considerablelatitude in selecting appropriate regions of genes for use in thepresent invention.

By “enzymatic RNA” is meant an RNA molecule with enzymatic activity(Cech, (1988) J. American. Med. Assoc. 260, 3030-3035). Enzymaticnucleic acids (ribozymes) act by first binding to a target RNA. Suchbinding occurs through the target binding portion of an enzymaticnucleic acid which is held in close proximity to an enzymatic portion ofthe molecule that acts to cleave the target RNA. Thus, the enzymaticnucleic acid first recognizes and then binds a target RNA through basepairing, and once bound to the correct site, acts enzymatically to cutthe target RNA.

By “decoy RNA” is meant an RNA molecule that mimics the natural bindingdomain for a ligand. The decoy RNA therefore competes with naturalbinding target for the binding of a specific ligand. For example, it hasbeen shown that over-expression of HIV trans-activation response (TAR)RNA can act as a “decoy” and efficiently binds HIV tat protein, therebypreventing it from binding to TAR sequences encoded in the HIV RNA. Thisis meant to be a specific example. Those in the art will recognize thatthis is but one example, and other embodiments can be readily generatedusing techniques generally known in the art.

As used herein, the term “monomers” typically indicates monomers linkedby phosphodiester bonds or analogs thereof to form oligonucleotidesranging in size from a few monomeric units, e.g., from about 3-4, toabout several hundreds of monomeric units. Analogs of phosphodiesterlinkages include: phosphorothioate, phosphorodithioate,methylphosphomates, phosphoroselenoate, phosphoramidate, and the like,as more fully described below.

The term “nucleotide” covers naturally occurring nucleotides as well asnonnaturally occurring nucleotides. It should be clear to the personskilled in the art that various nucleotides which previously have beenconsidered “non-naturally occurring” have subsequently been found innature. Thus, “nucleotides” includes not only the known purine andpyrimidine heterocycles-containing molecules, but also heterocyclicanalogues and tautomers thereof. Illustrative examples of other types ofnucleotides are molecules containing adenine, guanine, thymine,cytosine, uracil, purine, xanthine, diaminopurine,8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine,N4,N4-ethanocytosin, N6,N6-ethano-2,6-diaminopurine, 5-methylcytosine,5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine,isoguanin, inosine and the “non-naturally occurring” nucleotidesdescribed in Benner et al., U.S. Pat. No. 5,432,272. The term“nucleotide” is intended to cover every and all of these examples aswell as analogues and tautomers thereof. Especially interestingnucleotides are those containing adenine, guanine, thymine, cytosine,and uracil, which are considered as the naturally occurring nucleotidesin relation to therapeutic and diagnostic application in humans.Nucleotides include the natural 2′-deoxy and 2′-hydroxyl sugars, e.g.,as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman,San Francisco, 1992) as well as their analogs.

“Analogs” in reference to nucleotides includes synthetic nucleotideshaving modified base moieties and/or modified sugar moieties (see e.g.,described generally by Scheit, Nucleotide Analogs, John Wiley, New York,1980; Freier & Altmann, (1997) Nucl. Acid. Res., 25(22), 4429-4443,Toulme, J. J., (2001) Nature Biotechnology 19:17-18; Manoharan M.,(1999) Biochemica et Biophysica Acta 1489:117-139; Freier S. M., (1997)Nucleic Acid Research, 25:4429-4443, Uhlman, E., (2000) Drug Discovery &Development, 3: 203-213, Herdewin P., (2000) Antisense & Nucleic AcidDrug Dev., 10:297-310); 2′-O, 3′-C-linked[3.2.0]bicycloarabinonucleosides. Such analogs include syntheticnucleotides designed to enhance binding properties, e.g., duplex ortriplex stability, specificity, or the like.

As used herein, “hybridization” means the pairing of substantiallycomplementary strands of oligomeric compounds. One mechanism of pairinginvolves hydrogen bonding, which may be Watson-Crick, Hoogsteen orreversed Hoögsteen hydrogen bonding, between complementary nucleoside ornucleotide bases (nucleotides) of the strands of oligomeric compounds.For example, adenine and thymine are complementary nucleotides whichpair through the formation of hydrogen bonds. Hybridization can occurunder varying circumstances.

An antisense compound is “specifically hybridizable” when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a modulation of function and/oractivity, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo assays ortherapeutic treatment, and under conditions in which assays areperformed in the case of in vitro assays.

As used herein, the phrase “stringent hybridization conditions” or“stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated. In general, stringent hybridization conditionscomprise low concentrations (<0.15M) of salts with inorganic cationssuch as Na+ or K+ (i.e., low ionic strength), temperature higher than20° C.-25° C. below the Tm of the oligomeric compound:target sequencecomplex, and the presence of denaturants such as formamide,dimethylformamide, dimethyl sulfoxide, or the detergent sodium dodecylsulfate (SDS). For example, the hybridization rate decreases 1.1% foreach 1% formamide. An example of a high stringency hybridizationcondition is 0.1× sodium chloride-sodium citrate buffer (SSC)/0.1% (w/v)SDS at 60° C. for 30 minutes.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleotides on one or two oligomeric strands. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, said target nucleic acid being a DNA, RNA, oroligonucleotide molecule, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to be acomplementary position. The oligomeric compound and the further DNA,RNA, or oligonucleotide molecule are complementary to each other when asufficient number of complementary positions in each molecule areoccupied by nucleotides which can hydrogen bond with each other. Thus,“specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of precise pairing or complementarityover a sufficient number of nucleotides such that stable and specificbinding occurs between the oligomeric compound and a target nucleicacid.

It is understood in the art that the sequence of an oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure,mismatch or hairpin structure). The oligomeric compounds of the presentinvention comprise at least about 70%, or at least about 75%, or atleast about 80%, or at least about 85%, or at least about 90%, or atleast about 95%, or at least about 99% sequence complementarity to atarget region within the target nucleic acid sequence to which they aretargeted. For example, an antisense compound in which 18 of 20nucleotides of the antisense compound are complementary to a targetregion, and would therefore specifically hybridize, would represent 90percent complementarity. In this example, the remainingnon-complementary nucleotides may be clustered or interspersed withcomplementary nucleotides and need not be contiguous to each other or tocomplementary nucleotides. As such, an antisense compound which is 18nucleotides in length having 4 (four) non-complementary nucleotideswhich are flanked by two regions of complete complementarity with thetarget nucleic acid would have 77.8% overall complementarity with thetarget nucleic acid and would thus fall within the scope of the presentinvention. Percent complementarity of an antisense compound with aregion of a target nucleic acid can be determined routinely using BLASTprograms (basic local alignment search tools) and PowerBLAST programsknown in the art. Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., (1981) 2, 482-489).

As used herein, the term “Thermal Melting Point (Tm)” refers to thetemperature, under defined ionic strength, pH, and nucleic acidconcentration, at which 50% of the oligonucleotides complementary to thetarget sequence hybridize to the target sequence at equilibrium.Typically, stringent conditions will be those in which the saltconcentration is at least about 0.01 to 1.0 M Na ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short oligonucleotides (e.g., 10 to 50 nucleotide). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide.

As used herein, “modulation” means either an increase (stimulation) or adecrease (inhibition) in the expression of a gene.

The term “variant”, when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to a wild typegene. This definition may also include, for example, “allelic,”“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. Of particular utility in the invention are variants of wildtype gene products. Variants may result from at least one mutation inthe nucleic acid sequence and may result in altered mRNAs or inpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes that give rise to variants aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

The resulting polypeptides generally will have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between individuals ofa given species. Polymorphic variants also may encompass “singlenucleotide polymorphisms” (SNPs,) or single base mutations in which thepolynucleotide sequence varies by one base. The presence of SNPs may beindicative of, for example, a certain population with a propensity for adisease state, that is susceptibility versus resistance.

Derivative polynucleotides include nucleic acids subjected to chemicalmodification, for example, replacement of hydrogen by an alkyl, acyl, oramino group. Derivatives, e.g., derivative oligonucleotides, maycomprise non-naturally-occurring portions, such as altered sugarmoieties or inter-sugar linkages. Exemplary among these arephosphorothioate and other sulfur containing species which are known inthe art. Derivative nucleic acids may also contain labels, includingradionucleotides, enzymes, fluorescent agents, chemiluminescent agents,chromogenic agents, substrates, cofactors, inhibitors, magneticparticles, and the like.

A “derivative” polypeptide or peptide is one that is modified, forexample, by glycosylation, pegylation, phosphorylation, sulfation,reduction/alkylation, acylation, chemical coupling, or mild formalintreatment. A derivative may also be modified to contain a detectablelabel, either directly or indirectly, including, but not limited to, aradioisotope, fluorescent, and enzyme label.

As used herein, the term “animal” or “patient” is meant to include, forexample, humans, sheep, elks, deer, mule deer, minks, mammals, monkeys,horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken,reptiles, fish, insects and arachnids.

“Mammal” covers warm blooded mammals that are typically under medicalcare (e.g., humans and domesticated animals). Examples include feline,canine, equine, bovine, and human, as well as just human.

“Treating” or “treatment” covers the treatment of a disease-state in amammal, and includes: (a) preventing the disease-state from occurring ina mammal, in particular, when such mammal is predisposed to thedisease-state but has not yet been diagnosed as having it; (b)inhibiting the disease-state, e.g., arresting it development; and/or (c)relieving the disease-state, e.g., causing regression of the diseasestate until a desired endpoint is reached. Treating also includes theamelioration of a symptom of a disease (e.g., lessen the pain ordiscomfort), wherein such amelioration may or may not be directlyaffecting the disease (e.g., cause, transmission, expression, etc.).

As used herein a “Neurological disease or disorder” refers to anydisease or disorder of the nervous system and/or visual system.“Neurological disease or disorder” include disease or disorders thatinvolve the central nervous system (brain, brainstem and cerebellum),the peripheral nervous system (including cranial nerves), and theautonomic nervous system (parts of which are located in both central andperipheral nervous system). A Neurological disease or disorder includesbut is not limited to acquired epileptiform aphasia; acute disseminatedencephalomyelitis; adrenoleukodystrophy; age-related maculardegeneration; agenesis of the corpus callosum; agnosia; Aicardisyndrome; Alexander disease; Alpers' disease; alternating hemiplegia;Alzheimer's disease; Vascular dementia; amyotrophic lateral sclerosis;anencephaly; Angelman syndrome; angiomatosis; anoxia; aphasia; apraxia;arachnoid cysts; arachnoiditis; Anronl-Chiari malformation;arteriovenous malformation; Asperger syndrome; ataxia telegiectasia;attention deficit hyperactivity disorder; autism; autonomic dysfunction;back pain; Batten disease; Behcet's disease; Bell's palsy; benignessential blepharospasm; benign focal; amyotrophy; benign intracranialhypertension; Binswanger's disease; blepharospasm; Bloch Sulzbergersyndrome; brachial plexus injury; brain abscess; brain injury; braintumors (including glioblastoma multiforme); spinal tumor; Brown-Sequardsyndrome; Canavan disease; carpal tunnel syndrome; causalgia; centralpain syndrome; central pontine myelinolysis; cephalic disorder; cerebralaneurysm; cerebral arteriosclerosis; cerebral atrophy; cerebralgigantism; cerebral palsy; Charcot-Marie-Tooth disease;chemotherapy-induced neuropathy and neuropathic pain; Chiarimalformation; chorea; chronic inflammatory demyelinating polyneuropathy;chronic pain; chronic regional pain syndrome; Coffin Lowry syndrome;coma, including persistent vegetative state; congenital facial diplegia;corticobasal degeneration; cranial arteritis; craniosynostosis;Creutzfeldt-Jakob disease; cumulative trauma disorders; Cushing'ssyndrome; cytomegalic inclusion body disease; cytomegalovirus infection;dancing eyes-dancing feet syndrome; DandyWalker syndrome; Dawsondisease; De Morsier's syndrome; Dejerine-Klumke palsy; dementia;dermatomyositis; diabetic neuropathy; diffuse sclerosis; dysautonomia;dysgraphia; dyslexia; dystonias; early infantile epilepticencephalopathy; empty sella syndrome; encephalitis; encephaloceles;encephalotrigeminal angiomatosis; epilepsy; Erb's palsy; essentialtremor; Fabry's disease; Fahr's syndrome; fainting; familial spasticparalysis; febrile seizures; Fisher syndrome; Friedreich's ataxia;fronto-temporal dementia and other “tauopathies”; Gaucher's disease;Gerstmann's syndrome; giant cell arteritis; giant cell inclusiondisease; globoid cell leukodystrophy; Guillain-Barre syndrome;HTLV-1-associated myelopathy; Hallervorden-Spatz disease; head injury;headache; hemifacial spasm; hereditary spastic paraplegia; heredopathiaatactic a polyneuritiformis; herpes zoster oticus; herpes zoster;Hirayama syndrome; HIV associated dementia and neuropathy (alsoneurological manifestations of AIDS); holoprosencephaly; Huntington'sdisease and other polyglutamine repeat diseases; hydranencephaly;hydrocephalus; hypercortisolism; hypoxia; immune-mediatedencephalomyelitis; inclusion body myositis; incontinentia pigmenti;infantile phytanic acid storage disease; infantile refsum disease;infantile spasms; inflammatory myopathy; intracranial cyst; intracranialhypertension; Joubert syndrome; Keams-Sayre syndrome; Kennedy diseaseKinsboume syndrome; Klippel Feil syndrome; Krabbe disease;Kugelberg-Welander disease; kuru; Lafora disease; Lambert-Eatonmyasthenic syndrome; Landau-Kleffner syndrome; lateral medullary(Wallenberg) syndrome; learning disabilities; Leigh's disease;Lennox-Gustaut syndrome; Lesch-Nyhan syndrome; leukodystrophy; Lewy bodydementia; Lissencephaly; locked-in syndrome; Lou Gehrig's disease (i.e.,motor neuron disease or amyotrophic lateral sclerosis); lumbar discdisease; Lyme disease—neurological sequelae; Machado-Joseph disease;macrencephaly; megalencephaly; Mclkersson-Rosenthal syndrome; Menieresdisease; meningitis; Menkes disease; metachromatic leukodystrophy;microcephaly; migraine; Miller Fisher syndrome; mini-strokes;mitochondrial myopathies; Mobius syndrome; monomelic amyotrophy; motorneuron disease; Moyamoya disease; mucopolysaccharidoses; milti-infarctdementia; multifocal motor neuropathy; multiple sclerosis and otherdemyelinating disorders; multiple system atrophy with posturalhypotension; muscular dystrophy; myasthenia gravis; myelinoclasticdiffuse sclerosis; myoclonic encephalopathy of infants; myoclonus;myopathy; myotonia congenital; narcolepsy; neurofibromatosis;neuroleptic malignant syndrome; neurological manifestations of AIDS;neurological sequelae of lupus; neuromyotonia; neuronal ceroidlipofuscinosis; neuronal migration disorders; Niemann-Pick disease;O'Sullivan-McLeod syndrome; occipital neuralgia; occult spinaldysraphism sequence; Ohtahara syndrome; olivopontocerebellar atrophy;opsoclonus myoclonus; optic neuritis; orthostatic hypotension; overusesyndrome; paresthesia; a neurodegenerative disease or disorder(Parkinson's disease, Huntington's disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), dementia, multiple sclerosis andother diseases and disorders associated with neuronal cell death);paramyotonia congenital; paraneoplastic diseases; paroxysmal attacks;Parry Romberg syndrome; Pelizaeus-Merzbacher disease; periodicparalyses; peripheral neuropathy; painful neuropathy and neuropathicpain; persistent vegetative state; pervasive developmental disorders;photic sneeze reflex; phytanic acid storage disease; Pick's disease;pinched nerve; pituitary tumors; polymyositis; porencephaly; post-poliosyndrome; postherpetic neuralgia; postinfectious encephalomyelitis;postural hypotension; Prader-Willi syndrome; primary lateral sclerosis;prion diseases; progressive hemifacial atrophy; progressivemultifocalleukoencephalopathy; progressive sclerosing poliodystrophy;progressive supranuclear palsy; pseudotumor cerebri; Ramsay-Huntsyndrome (types I and 11); Rasmussen's encephalitis; reflex sympatheticdystrophy syndrome; Refsum disease; repetitive motion disorders;repetitive stress injuries; restless legs syndrome;retrovirus-associated myelopathy; Rett syndrome; Reye's syndrome; SaintVitus dance; Sandhoff disease; Schilder's disease; schizencephaly;septo-optic dysplasia; shaken baby syndrome; shingles; Shy-Dragersyndrome; Sjogren's syndrome; sleep apnea; Soto's syndrome; spasticity;spina bifida; spinal cord injury; spinal cord tumors; spinal muscularatrophy; Stiff-Person syndrome; stroke; Sturge-Weber syndrome; subacutesclerosing panencephalitis; subcortical arteriosclerotic encephalopathy;Sydenham chorea; syncope; syringomyelia; tardive dyskinesia; Tay-Sachsdisease; temporal arteritis; tethered spinal cord syndrome; Thomsendisease; thoracic outlet syndrome; Tic Douloureux; Todd's paralysis;Tourette syndrome; transient ischemic attack; transmissible spongiformencephalopathies; transverse myelitis; traumatic brain injury; tremor;trigeminal neuralgia; tropical spastic paraparesis; tuberous sclerosis;vascular dementia (multi-infarct dementia); vasculitis includingtemporal arteritis; Von Hippel-Lindau disease; Wallenberg's syndrome;Werdnig-Hoffman disease; West syndrome; whiplash; Williams syndrome;Wildon's disease; and Zellweger syndrome.

Polynucleotide and Oligonucleotide Compositions and Molecules

Targets:

In one embodiment, the targets comprise nucleic acid sequences ofAlpha-L-Iduronidase (IDUA), including without limitation sense and/orantisense noncoding and/or coding sequences associated with IDUA.

Alpha-L-Iduronidase (a-L-Iduronidase or a-L-iduronide iduronohydrolaseE.C.3.2.1.76; IDUA) is a lysosomal hydrolase required for the breakdownof the glycosaminoglycans heparin sulfate and dermatan sulfate.Lysosomal enzymes undergo a series of processing and maturation eventsfor which IDUA has served as a model.

The lysosomal hydrolase a-L-iduronidase (IDUA) is one of the enzymes inthe metabolic pathway responsible for the degradation of theglycosaminoglycans heparan sulfate and dermatan sulfate. In humans adeficiency of IDUA leads to the accumulation of glycosaminoglycans,resulting in the lysosomal storage disorder mucopolysaccharidosis typeI.

A genetic deficiency of the carbohydrate-cleaving, lysosomal enzyme.alpha.-L-iduronidase causes a lysosomal storage disorder known asmucopolysaccharidosis I (MPS I). In a severe form, MPS I is commonlyknown as Hurler syndrome and is associated with multiple problems suchas mental retardation, clouding of the cornea, coarsened facialfeatures, cardiac disease, respiratory disease, liver and spleenenlargement, hernias, and joint stiffness. Patients suffering fromHurler syndrome usually die before age 10. In an intermediate form knownas Hurler-Scheie syndrome, mental function is generally not severelyaffected, but physical problems may lead to death by the teens ortwenties. Scheie syndrome is the mildest form of MPS I. It is compatiblewith a normal life span, but joint stiffness, corneal clouding and heartvalve disease cause significant problems.

Type I mucopolysaccharidosis (MPS), also known as Hurler's syndrome, isan inherited metabolic disease caused by a defect in the enzyme.alpha.-L-iduronidase (IDUA), which functions to degrademucopolysaccharides. An insufficient level of IDUA causes a pathologicalbuildup of heparan sulfate and dermatan sulfate in, e.g., the heart,liver, and central nervous system. Symptoms including neurodegenerationand mental retardation appear during childhood and early death can occurdue to organ damage. Typically, treatment includes intravenous enzymereplacement therapy with recombinant IDUA. However, systemicallyadministered recombinant IDUA does not cross the blood brain barrier(BBB), and therefore has little impact on the effects of the disease inthe central nervous system (CNS).

In an embodiment, antisense oligonucleotides are used to prevent ortreat diseases or disorders associated with IDUA family members.Exemplary Alpha-L-Iduronidase (IDUA) mediated diseases and disorderswhich can be treated with the antisensense oligonucleotides of theinvention and/or with cell/tissues regenerated from stem cells obtainedusing and/or having the antisense compounds comprise: a disease ordisorder associated with abnormal function and/or expression ofAlpha-L-Iduronidase; Mucopolysaccharidosis I (MPS I); a disease ordisorder associated with abnormal levels of heparan sulfate and/ordermatan sulfate; a neurological disease or disorder, aneurodegenerative disease or disorder, long-term memory impairment,Hurler syndrome; Hurler-Scheie syndrome and Scheie syndrome etc.

In an embodiment, modulation of IDUA by one or more antisenseoligonucleotides is administered to a patient in need thereof, toprevent or treat any disease or disorder related to IDUA abnormalexpression, function, activity as compared to a normal control.

In an embodiment, the oligonucleotides are specific for polynucleotidesof IDUA, which includes, without limitation noncoding regions. The IDUAtargets comprise variants of IDUA; mutants of IDUA, including SNPs;noncoding sequences of IDUA; alleles, fragments and the like. Preferablythe oligonucleotide is an antisense RNA molecule.

In accordance with embodiments of the invention, the target nucleic acidmolecule is not limited to IDUA polynucleotides alone but extends to anyof the isoforms, receptors, homologs, non-coding regions and the like ofIDUA.

In an embodiment, an oligonucleotide targets a natural antisensesequence (natural antisense to the coding and non-coding regions) ofIDUA targets, including, without limitation, variants, alleles,homologs, mutants, derivatives, fragments and complementary sequencesthereto. Preferably the oligonucleotide is an antisense RNA or DNAmolecule.

In an embodiment, the oligomeric compounds of the present invention alsoinclude variants in which a different base is present at one or more ofthe nucleotide positions in the compound. For example, if the firstnucleotide is an adenine, variants may be produced which containthymidine, guanosine, cytidine or other natural or unnatural nucleotidesat this position. This may be done at any of the positions of theantisense compound. These compounds are then tested using the methodsdescribed herein to determine their ability to inhibit expression of atarget nucleic acid.

In some embodiments, homology, sequence identity or complementarity,between the antisense compound and target is from about 50% to about60%. In some embodiments, homology, sequence identity orcomplementarity, is from about 60% to about 70%. In some embodiments,homology, sequence identity or complementarity, is from about 70% toabout 80%. In some embodiments, homology, sequence identity orcomplementarity, is from about 80% to about 90%. In some embodiments,homology, sequence identity or complementarity, is about 90%, about 92%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100%.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired. Such conditionsinclude, i.e., physiological conditions in the case of in vivo assays ortherapeutic treatment, and conditions in which assays are performed inthe case of in vitro assays.

An antisense compound, whether DNA, RNA, chimeric, substituted etc, isspecifically hybridizable when binding of the compound to the target DNAor RNA molecule interferes with the normal function of the target DNA orRNA to cause a loss of utility, and there is a sufficient degree ofcomplementarily to avoid non-specific binding of the antisense compoundto non-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed.

In an embodiment, targeting of IDUA including without limitation,antisense sequences which are identified and expanded, using forexample, PCR, hybridization etc., one or more of the sequences set forthas SEQ ID NOS: 2 to 9, and the like, modulate the expression or functionof IDUA. In one embodiment, expression or function is up-regulated ascompared to a control. In an embodiment, expression or function isdown-regulated as compared to a control.

In an embodiment, oligonucleotides comprise nucleic acid sequences setforth as SEQ ID NOS: 10 to 28 including antisense sequences which areidentified and expanded, using for example, PCR, hybridization etc.These oligonucleotides can comprise one or more modified nucleotides,shorter or longer fragments, modified bonds and the like. Examples ofmodified bonds or internucleotide linkages comprise phosphorothioate,phosphorodithioate or the like. In an embodiment, the nucleotidescomprise a phosphorus derivative. The phosphorus derivative (or modifiedphosphate group) which may be attached to the sugar or sugar analogmoiety in the modified oligonucleotides of the present invention may bea monophosphate, diphosphate, triphosphate, alkylphosphate,alkanephosphate, phosphorothioate and the like. The preparation of theabove-noted phosphate analogs, and their incorporation into nucleotides,modified nucleotides and oligonucleotides, per se, is also known andneed not be described here.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotides have been safelyand effectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

In embodiments of the present invention oligomeric antisense compounds,particularly oligonucleotides, bind to target nucleic acid molecules andmodulate the expression and/or function of molecules encoded by a targetgene. The functions of DNA to be interfered comprise, for example,replication and transcription. The functions of RNA to be interferedcomprise all vital functions such as, for example, translocation of theRNA to the site of protein translation, translation of protein from theRNA, splicing of the RNA to yield one or more mRNA species, andcatalytic activity which may be engaged in or facilitated by the RNA.The functions may be up-regulated or inhibited depending on thefunctions desired.

The antisense compounds, include, antisense oligomeric compounds,antisense oligonucleotides, external guide sequence (EGS)oligonucleotides, alternate splicers, primers, probes, and otheroligomeric compounds that hybridize to at least a portion of the targetnucleic acid. As such, these compounds may be introduced in the form ofsingle-stranded, double-stranded, partially single-stranded, or circularoligomeric compounds.

Targeting an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes Alpha-L-Iduronidase (IDUA).

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

In an embodiment, the antisense oligonucleotides bind to the naturalantisense sequences of Alpha-L-Iduronidase (IDUA) and modulate theexpression and/or function of IDUA (SEQ ID NO: 1). Examples of antisensesequences include SEQ ID NOS: 2 to 28.

In an embodiment, the antisense oligonucleotides bind to one or moresegments of Alpha-L-Iduronidase (IDUA) polynucleotides and modulate theexpression and/or function of IDUA. The segments comprise at least fiveconsecutive nucleotides of the IDUA sense or antisense polynucleotides.

In an embodiment, the antisense oligonucleotides are specific fornatural antisense sequences of IDUA wherein binding of theoligonucleotides to the natural antisense sequences of IDUA modulateexpression and/or function of IDUA.

In an embodiment, oligonucleotide compounds comprise sequences set forthas SEQ ID NOS: 10 to 28, antisense sequences which are identified andexpanded, using for example, PCR, hybridization etc Theseoligonucleotides can comprise one or more modified nucleotides, shorteror longer fragments, modified bonds and the like. Examples of modifiedbonds or internucleotide linkages comprise phosphorothioate,phosphorodithioate or the like. In an embodiment, the nucleotidescomprise a phosphorus derivative. The phosphorus derivative (or modifiedphosphate group) which may be attached to the sugar or sugar analogmoiety in the modified oligonucleotides of the present invention may bea monophosphate, diphosphate, triphosphate, alkylphosphate,alkanephosphate, phosphorothioate and the like. The preparation of theabove-noted phosphate analogs, and their incorporation into nucleotides,modified nucleotides and oligonucleotides, per se, is also known andneed not be described here.

Since, as is known in the art, the translation initiation codon istypically 9-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes has a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG; and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes).Eukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or wider a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA transcribed from a geneencoding Alpha-L-Iduronidase (IDUA), regardless of the sequence(s) ofsuch codons. A translation termination codon (or “stop codon”) of a genemay have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (thecorresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA,respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions that may betargeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, atargeted region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Another target region includes the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene). Still another target regionincludes the 3′ untranslated region (3′UTR), known in the art to referto the portion of an mRNA in the 3′ direction from the translationtermination codon, and thus including nucleotides between thetranslation termination codon and 3′ end of an mRNA (or correspondingnucleotides on the gene). The 5′ cap site of an mRNA comprises anN7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap site. Another target region for thisinvention is the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are exercisedfrom a transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. In one embodiment, targeting splicesites, i.e., intron-exon junctions or exon-intron junctions, isparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular splice product isimplicated in disease. An aberrant fusion junction due to rearrangementor deletion is another embodiment of a target site. mRNA transcriptsproduced via the process of splicing of two (or more) mRNAs fromdifferent gene sources are known as “fusion transcripts”. Introns can beeffectively targeted using antisense compounds targeted to, for example,DNA or pre-mRNA.

In an embodiment, the antisense oligonucleotides bind to coding and/ornon-coding regions of a target polynucleotide and modulate theexpression and/or function of the target molecule.

In an embodiment, the antisense oligonucleotides bind to naturalantisense polynucleotides and modulate the expression and/or function ofthe target molecule.

In an embodiment, the antisense oligonucleotides bind to sensepolynucleotides and modulate the expression and/or function of thetarget molecule.

Alternative RNA transcripts can be produced from the same genomic regionof DNA. These alternative transcripts are generally known as “variants”.More specifically, “pre-mRNA variants” are transcripts produced from thesame genomic DNA that differ from other transcripts produced from thesame genomic DNA in either their start or stop position and contain bothintronic and exonic sequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

Variants can be produced through the use of alternative signals to startor stop transcription. Pre-mRNAs and mRNAs can possess more than onestart codon or stop codon. Variants that originate from a pre-mRNA ormRNA that use alternative start codons are known as “alternative startvariants” of that pre-mRNA or mRNA. Those transcripts that use analternative stop codon are known as “alternative stop variants” of thatpre-mRNA or mRNA. One specific type of alternative stop variant is the“polyA variant” in which the multiple transcripts produced result fromthe alternative selection of one of the “polyA stop signals” by thetranscription machinery, thereby producing transcripts that terminate atunique polyA sites. Within the context of the invention, the types ofvariants described herein are also embodiments of target nucleic acids.

The locations on the target nucleic acid to which the antisensecompounds hybridize are defined as at least a 5-nucleotide long portionof a target region to which an active antisense compound is targeted.

While the specific sequences of certain exemplary target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional target segments are readilyidentifiable by one having ordinary skill in the art in view of thisdisclosure.

Target segments 5-100 nucleotides in length comprising a stretch of atleast five (5) consecutive nucleotides selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

Target segments can include DNA or RNA sequences that comprise at leastthe 5 consecutive nucleotides from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleotides beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 5 to about 100 nucleotides). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 5 consecutive nucleotides from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleotides being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 5 to about 100nucleotides). One having skill in the art armed with the target segmentsillustrated herein will be able, without undue experimentation, toidentify further preferred target segments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

In embodiments of the invention the oligonucleotides bind to anantisense strand of a particular target. The oligonucleotides are atleast 5 nucleotides in length and can be synthesized so eacholigonucleotide targets overlapping sequences such that oligonucleotidesare synthesized to cover the entire length of the target polynucleotide.The targets also include coding as well as non coding regions.

In one embodiment, it is preferred to target specific nucleic acids byantisense oligonucleotides. Targeting an antisense compound to aparticular nucleic acid is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a non coding polynucleotidesuch as for example, non coding RNA (ncRNA).

RNAs can be classified into (1) messenger RNAs (mRNAs), which aretranslated into proteins, and (2) non-protein-coding RNAs (ncRNAs).ncRNAs comprise microRNAs, antisense transcripts and otherTranscriptional Units (TU) containing a high density of stop codons andlacking any extensive “Open Reading Frame”. Many ncRNAs appear to startfrom initiation sites in 3′ untranslated regions (3′UTRs) ofprotein-coding loci. ncRNAs are often rare and at least half of thencRNAs that have been sequenced by the FANTOM consortium seem not to bepolyadenylated. Most researchers have for obvious reasons focused onpolyadenylated mRNAs that are processed and exported to the cytoplasm.Recently, it was shown that the set of non-polyadenylated nuclear RNAsmay be very large, and that many such transcripts arise from so-calledintergenic regions. The mechanism by which ncRNAs may regulate geneexpression is by base pairing with target transcripts. The RNAs thatfunction by base pairing can be grouped into (1) cis encoded RNAs thatare encoded at the same genetic location, but on the opposite strand tothe RNAs they act upon and therefore display perfect complementarity totheir target, and (2) trans-encoded RNAs that are encoded at achromosomal location distinct from the RNAs they act upon and generallydo not exhibit perfect base-pairing potential with their targets.

Without wishing to be bound by theory, perturbation of an antisensepolynucleotide by the antisense oligonucleotides described herein canalter the expression of the corresponding sense messenger RNAs. However,this regulation can either be discordant (antisense knockdown results inmessenger RNA elevation) or concordant (antisense knockdown results inconcomitant messenger RNA reduction). In these cases, antisenseoligonucleotides can be targeted to overlapping or non-overlapping partsof the antisense transcript resulting in its knockdown or sequestration.Coding as well as non-coding antisense can be targeted in an identicalmanner and that either category is capable of regulating thecorresponding sense transcripts—either in a concordant or disconcordantmanner. The strategies that are employed in identifying newoligonucleotides for use against a target can be based on the knockdownof antisense RNA transcripts by antisense oligonucleotides or any othermeans of modulating the desired target.

Strategy 1:

In the case of discordant regulation, knocking down the antisensetranscript elevates the expression of the conventional (sense) gene.Should that latter gene encode for a known or putative drug target, thenknockdown of its antisense counterpart could conceivably mimic theaction of a receptor agonist or an enzyme stimulant.

Strategy 2:

In the case of concordant regulation, one could concomitantly knock downboth antisense and sense transcripts and thereby achieve synergisticreduction of the conventional (sense) gene expression. If, for example,an antisense oligonucleotide is used to achieve knockdown, then thisstrategy can be used to apply one antisense oligonucleotide targeted tothe sense transcript and another antisense oligonucleotide to thecorresponding antisense transcript, or a single energetically symmetricantisense oligonucleotide that simultaneously targets overlapping senseand antisense transcripts.

According to the present invention, antisense compounds includeantisense oligonucleotides, ribozymes, external guide sequence (EGS)oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, and otheroligomeric compounds which hybridize to at least a portion of the targetnucleic acid and modulate its function. As such, they may be DNA, RNA,DNA-like, RNA-like, or mixtures thereof, or may be mimetics of one ormore of these. These compounds may be single-stranded, doublestranded,circular or hairpin oligomeric compounds and may contain structuralelements such as internal or terminal bulges, mismatches or loops.Antisense compounds are routinely prepared linearly but can be joined orotherwise prepared to be circular and/or branched. Antisense compoundscan include constructs such as, for example, two strands hybridized toform a wholly or partially double-stranded compound or a single strandwith sufficient self-complementarity to allow for hybridization andformation of a fully or partially double-stranded compound. The twostrands can be linked internally leaving free 3′ or 5′ termini or can belinked to form a continuous hairpin structure or loop. The hairpinstructure may contain an overhang on either the 5′ or 3′ terminusproducing an extension of single stranded character. The double strandedcompounds optionally can include overhangs on the ends. Furthermodifications can include conjugate groups attached to one of thetermini, selected nucleotide positions, sugar positions or to one of theinternucleoside linkages. Alternatively, the two strands can be linkedvia a non-nucleic acid moiety or linker group. When formed from only onestrand, dsRNA can take the form of a self-complementary hairpin-typemolecule that doubles back on itself to form a duplex. Thus, the dsRNAscan be fully or partially double stranded. Specific modulation of geneexpression can be achieved by stable expression of dsRNA hairpins intransgenic cell lines, however, in some embodiments, the gene expressionor function is up regulated. When formed from two strands, or a singlestrand that takes the form of a self-complementary hairpin-type moleculedoubled back on itself to form a duplex, the two strands (orduplex-forming regions of a single strand) are complementary RNA strandsthat base pair in Watson-Crick fashion.

Once introduced to a system, the compounds of the invention may elicitthe action of one or more enzymes or structural proteins to effectcleavage or other modification of the target nucleic acid or may workvia occupancy-based mechanisms. In general, nucleic acids (includingoligonucleotides) may be described as “DNA-like” (i.e., generally havingone or more 2′-deoxy sugars and, generally, T rather than U bases) or“RNA-like” (i.e., generally having one or more 2′-hydroxyl or2′-modified sugars and, generally U rather than T bases). Nucleic acidhelices can adopt more than one type of structure, most commonly the A-and B-forms. It is believed that, in general, oligonucleotides whichhave B-form-like structure are “DNA-like” and those which haveA-formlike structure are “RNA-like.” In some (chimeric) embodiments, anantisense compound may contain both A- and B-form regions.

In an embodiment, the desired oligonucleotides or antisense compounds,comprise at least one of: antisense RNA, antisense DNA, chimericantisense oligonucleotides, antisense oligonucleotides comprisingmodified linkages, interference RNA (RNAi), short interfering RNA(siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA(stRNA); or a short, hairpin RNA (shRNA); small RNA-induced geneactivation (RNAa); small activating RNAs (saRNAs), or combinationsthereof.

dsRNA can also activate gene expression, a mechanism that has beentermed “small RNA-induced gene activation” or RNAa. dsRNAs targetinggene promoters induce potent transcriptional activation of associatedgenes. RNAa was demonstrated in human cells using synthetic dsRNAs,termed “small activating RNAs” (saRNAs). It is currently not knownwhether RNAa is conserved in other organisms.

Small double-stranded RNA (dsRNA), such as small interfering RNA (siRNA)and microRNA (miRNA), have been found to be the trigger of anevolutionary conserved mechanism known as RNA interference (RNAi). RNAiinvariably leads to gene silencing via remodeling chromatin to therebysuppress transcription, degrading complementary mRNA, or blockingprotein translation. However, in instances described in detail in theexamples section which follows, oligonucleotides are shown to increasethe expression and/or function of the Alpha-L-Iduronidase (IDUA)polynucleotides and encoded products thereof. dsRNAs may also act assmall activating RNAs (saRNA). Without wishing to be bound by theory, bytargeting sequences in gene promoters, saRNAs would induce target geneexpression in a phenomenon referred to as dsRNA-induced transcriptionalactivation (RNAa).

In a further embodiment, the “preferred target segments” identifiedherein may be employed in a screen for additional compounds thatmodulate the expression of Alpha-L-Iduronidase (IDUA) polynucleotides.“Modulators” are those compounds that decrease or increase theexpression of a nucleic acid molecule encoding IDUA and which compriseat least a 5-nucleotide portion that is complementary to a preferredtarget segment. The screening method comprises the steps of contacting apreferred target segment of a nucleic acid molecule encoding sense ornatural antisense polynucleotides of IDUA with one or more candidatemodulators, and selecting for one or more candidate modulators whichdecrease or increase the expression of a nucleic acid molecule encodingIDUA polynucleotides, e.g. SEQ ID NOS: 10 to 28. Once it is shown thatthe candidate modulator or modulators are capable of modulating (e.g.either decreasing or increasing) the expression of a nucleic acidmolecule encoding IDUA polynucleotides, the modulator may then beemployed in further investigative studies of the function of IDUApolynucleotides, or for use as a research, diagnostic, or therapeuticagent in accordance with the present invention.

Targeting the natural antisense sequence preferably modulates thefunction of the target gene. For example, the IDUA gene (e.g. accessionnumber NM_(—)000203). In an embodiment, the target is an antisensepolynucleotide of the IDUA gene. In an embodiment, an antisenseoligonucleotide targets sense and/or natural antisense sequences of IDUApolynucleotides (e.g. accession number NM_(—)000203), variants, alleles,isoforms, homologs, mutants, derivatives, fragments and complementarysequences thereto. Preferably the oligonucleotide is an antisensemolecule and the targets include coding and noncoding regions ofantisense and/or sense IDUA polynucleotides.

The preferred target segments of the present invention may be also becombined with their respective complementary antisense compounds of thepresent invention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocessing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications. For example, suchdouble-stranded moieties have been shown to inhibit the target by theclassical hybridization of antisense strand of the duplex to the target,thereby triggering enzymatic degradation of the target.

In an embodiment, an antisense oligonucleotide targetsAlpha-L-Iduronidase (IDUA) polynucleotides (e.g. accession numberNM_(—)000203), variants, alleles, homologs, mutants, derivatives,fragments and complementary sequences thereto. Preferably theoligonucleotide is an antisense molecule.

In accordance with embodiments of the invention, the target nucleic acidmolecule is not limited to IDUA alone but extends to any polynucleotidevariant thereof and any polynucleotide that produces, affects, impactsor results in or relates to an IDUA expression product and/or anyisoforms thereof.

In an embodiment, an oligonucleotide targets a natural antisensesequence of IDUA polynucleotides, for example, polynucleotides set forthas SEQ ID NOS: 2 to 9, and any variants, alleles, homologs, mutants,derivatives, fragments and complementary sequences thereto. Examples ofantisense oligonucleotides are set forth as SEQ ID NOS: 10 to 28.

In one embodiment, the oligonucleotides are complementary to or bind tonucleic acid sequences of IDUA antisense, including without limitationnoncoding sense and/or antisense sequences associated with IDUApolynucleotides and modulate expression and/or function of IDUAmolecules.

In an embodiment, the oligonucleotides are complementary to or bind tonucleic acid sequences of IDUA natural antisense, set forth as SEQ IDNOS: 2 to 9, and modulate expression and/or function of IDUA molecules.

In an embodiment, oligonucleotides comprise sequences of at least 5consecutive nucleotides of SEQ ID NOS: 10 to 28 and modulate expressionand/or function of IDUA molecules.

The polynucleotide targets comprise IDUA, including family membersthereof, variants of IDUA; mutants of IDUA, including SNPs; noncodingsequences of IDUA; alleles of IDUA; species variants, fragments and thelike. Preferably the oligonucleotide is an antisense molecule.

In an embodiment, the oligonucleotide targeting IDUA polynucleotides,comprise: antisense RNA, interference RNA (RNAi), short interfering RNA(siRNA); micro interfering RNA (miRNA); a small, temporal RNA (stRNA);or a short, hairpin RNA (shRNA); small RNA-induced gene activation(RNAa); or, small activating RNA (saRNA).

In an embodiment, targeting of Alpha-L-Iduronidase (IDUA)polynucleotides, e.g. SEQ ID NOS: 2 to 28 modulate the expression orfunction of these targets. In one embodiment, expression or function isup-regulated as compared to a control. In an embodiment, expression orfunction is down-regulated as compared to a control.

In an embodiment, antisense compounds comprise sequences set forth asSEQ ID NOS: 10 to 28. These oligonucleotides can comprise one or moremodified nucleotides, shorter or longer fragments, modified bonds andthe like.

In an embodiment, SEQ ID NOS: 10 to 28 comprise one or more LNAnucleotides. Table 1 shows exemplary antisense oligonucleotides usefulin the methods of the invention.

TABLE 1 Antisense Sequence ID Sequence Name Sequence SEQ ID NO: 10CUR-1820 T*C*T*C*T*C*G*C*C*T*T*T*C*C*C*T*C*C*C*T SEQ ID NO: 11 CUR-1821C*T*C*A*A*G*C*A*A*T*C*T*C*C*C*A*C*C*T*C*A SEQ ID NO: 12 CUR-1822T*C*C*C*A*G*C*T*A*C*T*C*A*G*G*A*G*G*C*T SEQ ID NO: 13 CUR-1823C*A*T*G*T*C*T*T*G*T*G*T*G*G*C*T*G*G*G*A*T SEQ ID NO: 14 CUR-1973G*A*G*T*C*A*T*C*G*G*T*C*C*T*C*A*G*A*G*C*A*G SEQ ID NO: 15 CUR-1975A*T*T*C*T*C*C*T*T*C*C*T*G*C*T*A*A*A*G*C SEQ ID NO: 16 CUR-1976A*T*T*A*T*T*T*C*G*T*A*T*T*G*C*T*T*T*G*G*C SEQ ID NO: 17 CUR-1978C*A*C*A*C*A*T*G*C*A*T*A*C*A*T*G*G*A*C*T SEQ ID NO: 18 CUR-1981C*T*C*A*G*T*T*C*T*C*T*G*A*C*G*C*T*T*T*G*A*G SEQ ID NO: 19 CUR-1984G*C*C*A*C*A*G*T*G*T*G*A*G*G*A*A*C*G SEQ ID NO: 20 CUR-1985G*T*A*A*T*A*A*T*T*T*T*T*C*C*T*G*A*C*C*C SEQ ID NO: 21 CUR-1987A*G*T*C*G*T*T*T*A*A*T*A*A*T*T*C*T*G*G*A*G*T SEQ ID NO: 22 CUR-1988T*T*A*C*T*A*A*G*T*T*T*C*A*T*G*A*G*G*T*T SEQ ID NO: 23 CUR-1974A*T*G*G*C*T*C*A*A*C*T*C*A*C*A*T*A*G*C*A SEQ ID NO: 24 CUR-1977T*T*A*T*A*C*A*A*T*G*T*T*T*G*C*T*T*G*G*A*T*T SEQ ID NO: 25 CUR-1986T*T*G*T*T*G*C*A*C*A*A*T*G*T*A*C*A*A*G SEQ ID NO: 26 CUR-1983T*G*G*T*T*G*C*T*C*T*C*A*G*G*A*G*G*C*G*G*C*T SEQ ID NO: 27 CUR-1979A*T*T*T*T*A*G*T*T*G*T*T*T*T*C*T*C*T*G*G SEQ ID NO: 28 CUR-1982C*A*C*G*G*T*G*T*G*G*G*A*C*T*G*G*T*G*G*T

The modulation of a desired target nucleic acid can be carried out inseveral ways known in the art. For example, antisense oligonucleotides,siRNA etc. Enzymatic nucleic acid molecules (e.g., ribozymes) arenucleic acid molecules capable of catalyzing one or more of a variety ofreactions, including the ability to repeatedly cleave other separatenucleic acid molecules in a nucleotide base sequence-specific manner.Such enzymatic nucleic acid molecules can be used, for example, totarget virtually any RNA transcript.

Because of their sequence-specificity, trans-cleaving enzymatic nucleicacid molecules show promise as therapeutic agents for human disease.Enzymatic nucleic acid molecules can be designed to cleave specific RNAtargets within the background of cellular RNA. Such a cleavage eventrenders the mRNA non-functional and abrogates protein expression fromthat RNA. In this manner, synthesis of a protein associated with adisease state can be selectively inhibited.

In general, enzymatic nucleic acids with RNA cleaving activity act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of an enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

Several approaches such as in vitro selection (evolution) strategies(Orgel, (1979) Proc. R. Soc. London, B 205, 435) have been used toevolve new nucleic acid catalysts capable of catalyzing a variety ofreactions, such as cleavage and ligation of phosphodiester linkages andamide linkages.

The development of ribozymes that are optimal for catalytic activitywould contribute significantly to any strategy that employs RNA-cleavingribozymes for the purpose of regulating gene expression. The hammerheadribozyme, for example, functions with a catalytic rate (kcat) of about 1min-1 in the presence of saturating (10 mM) concentrations of Mg2+cofactor. An artificial “RNA ligase” ribozyme has been shown to catalyzethe corresponding self-modification reaction with a rate of about 100min-1. In addition, it is known that certain modified hammerheadribozymes that have substrate binding arms made of DNA catalyze RNAcleavage with multiple turn-over rates that approach 100 min-1. Finally,replacement of a specific residue within the catalytic core of thehammerhead with certain nucleotide analogues gives modified ribozymesthat show as much as a 10-fold improvement in catalytic rate. Thesefindings demonstrate that ribozymes can promote chemical transformationswith catalytic rates that are significantly greater than those displayedin vitro by most natural self-cleaving ribozymes. It is then possiblethat the structures of certain selfcleaving ribozymes may be optimizedto give maximal catalytic activity, or that entirely new RNA motifs canbe made that display significantly faster rates for RNA phosphodiestercleavage.

Intermolecular cleavage of an RNA substrate by an RNA catalyst that fitsthe “hammerhead” model was first shown in 1987 (Uhlenbeck, O. C. (1987)Nature, 328: 596-600). The RNA catalyst was recovered and reacted withmultiple RNA molecules, demonstrating that it was truly catalytic.

Catalytic RNAs designed based on the “hammerhead” motif have been usedto cleave specific target sequences by making appropriate base changesin the catalytic RNA to maintain necessary base pairing with the targetsequences. This has allowed use of the catalytic RNA to cleave specifictarget sequences and indicates that catalytic RNAs designed according tothe “hammerhead” model may possibly cleave specific substrate RNAs invivo.

RNA interference (RNAi) has become a powerful tool for modulating geneexpression in mammals and mammalian cells. This approach requires thedelivery of small interfering RNA (siRNA) either as RNA itself or asDNA, using an expression plasmid or virus and the coding sequence forsmall hairpin RNAs that are processed to siRNAs. This system enablesefficient transport of the pre-siRNAs to the cytoplasm where they areactive and permit the use of regulated and tissue specific promoters forgene expression.

In an embodiment, an oligonucleotide or antisense compound comprises anoligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleicacid (DNA), or a mimetic, chimera, analog or homolog thereof. This termincludes oligonucleotides composed of naturally occurring nucleotides,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftendesired over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid and increased stability in the presence of nucleases.

According to the present invention, the oligonucleotides or “antisensecompounds” include antisense oligonucleotides (e.g. RNA, DNA, mimetic,chimera, analog or homolog thereof), ribozymes, external guide sequence(EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, saRNA, aRNA, andother oligomeric compounds which hybridize to at least a portion of thetarget nucleic acid and modulate its function. As such, they may be DNA,RNA, DNA-like, RNA-like, or mixtures thereof, or may be mimetics of oneor more of these. These compounds may be single-stranded,double-stranded, circular or hairpin oligomeric compounds and maycontain structural elements such as internal or terminal bulges,mismatches or loops. Antisense compounds are routinely prepared linearlybut can be joined or otherwise prepared to be circular and/or branched.Antisense compounds can include constructs such as, for example, twostrands hybridized to form a wholly or partially double-strandedcompound or a single strand with sufficient self-complementarity toallow for hybridization and formation of a fully or partiallydouble-stranded compound. The two strands can be linked internallyleaving free 3′ or 5′ termini or can be linked to form a continuoushairpin structure or loop. The hairpin structure may contain an overhangon either the 5′ or 3′ terminus producing an extension of singlestranded character. The double stranded compounds optionally can includeoverhangs on the ends. Further modifications can include conjugategroups attached to one of the termini, selected nucleotide positions,sugar positions or to one of the internucleoside linkages.Alternatively, the two strands can be linked via a non-nucleic acidmoiety or linker group. When formed from only one strand, dsRNA can takethe form of a self-complementary hairpin-type molecule that doubles backon itself to form a duplex. Thus, the dsRNAs can be fully or partiallydouble stranded. Specific modulation of gene expression can be achievedby stable expression of dsRNA hairpins in transgenic cell lines. Whenformed from two strands, or a single strand that takes the form of aself-complementary hairpin-type molecule doubled back on itself to forma duplex, the two strands (or duplex-forming regions of a single strand)are complementary RNA strands that base pair in Watson-Crick fashion.

Once introduced to a system, the compounds of the invention may elicitthe action of one or more enzymes or structural proteins to effectcleavage or other modification of the target nucleic acid or may workvia occupancy-based mechanisms. In general, nucleic acids (includingoligonucleotides) may be described as “DNA-like” (i.e., generally havingone or more 2′-deoxy sugars and, generally, T rather than U bases) or“RNA-like” (i.e., generally having one or more 2′-hydroxyl or2′-modified sugars and, generally U rather than T bases). Nucleic acidhelices can adopt more than one type of structure, most commonly the A-and B-forms. It is believed that, in general, oligonucleotides whichhave B-form-like structure are “DNA-like” and those which haveA-formlike structure are “RNA-like.” In some (chimeric) embodiments, anantisense compound may contain both A- and B-form regions.

The antisense compounds in accordance with this invention can comprisean antisense portion from about 5 to about 80 nucleotides (i.e. fromabout 5 to about 80 linked nucleosides) in length. This refers to thelength of the antisense strand or portion of the antisense compound. Inother words, a single-stranded antisense compound of the inventioncomprises from 5 to about 80 nucleotides, and a double-strandedantisense compound of the invention (such as a dsRNA, for example)comprises a sense and an antisense strand or portion of 5 to about 80nucleotides in length. One of ordinary skill in the art will appreciatethat this comprehends antisense portions of 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides inlength, or any range therewithin.

In one embodiment, the antisense compounds of the invention haveantisense portions of 10 to 50 nucleotides in length. One havingordinary skill in the art will appreciate that this embodiesoligonucleotides having antisense portions of 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleotides in length, or any range therewithin. In some embodiments,the oligonucleotides are 15 nucleotides in length.

In one embodiment, the antisense or oligonucleotide compounds of theinvention have antisense portions of 12 or 13 to 30 nucleotides inlength. One having ordinary skill in the art will appreciate that thisembodies antisense compounds having antisense portions of 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleotides in length, or any range therewithin.

In an embodiment, the oligomeric compounds of the present invention alsoinclude variants in which a different base is present at one or more ofthe nucleotide positions in the compound. For example, if the firstnucleotide is an adenosine, variants may be produced which containthymidine, guanosine or cytidine at this position. This may be done atany of the positions of the antisense or dsRNA compounds. Thesecompounds are then tested using the methods described herein todetermine their ability to inhibit expression of a target nucleic acid.

In some embodiments, homology, sequence identity or complementarity,between the antisense compound and target is from about 40% to about60%. In some embodiments, homology, sequence identity orcomplementarity, is from about 60% to about 70%. In some embodiments,homology, sequence identity or complementarity, is from about 70% toabout 80%. In some embodiments, homology, sequence identity orcomplementarity, is from about 80% to about 90%. In some embodiments,homology, sequence identity or complementarity, is about 90%, about 92%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100%.

In an embodiment, the antisense oligonucleotides, such as for example,nucleic acid molecules set forth in SEQ ID NOS: 10 to 28 comprise one ormore substitutions or modifications. In one embodiment, the nucleotidesare substituted with locked nucleic acids (LNA).

In an embodiment, the oligonucleotides target one or more regions of thenucleic acid molecules sense and/or antisense of coding and/ornon-coding sequences associated with IDUA and the sequences set forth asSEQ ID NOS: 1 to 9. The oligonucleotides are also targeted tooverlapping regions of SEQ ID NOS: 1 to 9.

Certain preferred oligonucleotides of this invention are chimericoligonucleotides. “Chimeric oligonucleotides” or “chimeras,” in thecontext of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionof modified nucleotides that confers one or more beneficial properties(such as, for example, increased nuclease resistance, increased uptakeinto cells, increased binding affinity for the target) and a region thatis a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of antisense modulation of gene expression.Consequently, comparable results can often be obtained with shorteroligonucleotides when chimeric oligonucleotides are used, compared tophosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art. In one an embodiment, a chimericoligonucleotide comprises at least one region modified to increasetarget binding affinity, and, usually, a region that acts as a substratefor RNAse H. Affinity of an oligonucleotide for its target (in thiscase, a nucleic acid encoding ras) is routinely determined by measuringthe Tm of an oligonucleotide/target pair, which is the temperature atwhich the oligonucleotide and target dissociate; dissociation isdetected spectrophotometrically. The higher the Tm, the greater is theaffinity of the oligonucleotide for the target.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotides mimetics as described above.Such; compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures comprise, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference.

In an embodiment, the region of the oligonucleotide which is modifiedcomprises at least one nucleotide modified at the 2′ position of thesugar, most preferably a 2′-Oalkyl, 2′-O-alkyl-O-alkyl or2′-fluoro-modified nucleotide. In other an embodiment, RNA modificationsinclude 2′-fluoro, 2′-amino and 2′ O-methyl modifications on the riboseof pyrimidines, abasic residues or an inverted base at the 3′ end of theRNA. Such modifications are routinely incorporated into oligonucleotidesand these oligonucleotides have been shown to have a higher Tm (i.e.,higher target binding affinity) than; 2′-deoxyoligonucleotides against agiven target. The effect of such increased affinity is to greatlyenhance RNAi oligonucleotide inhibition of gene expression. RNAse H is acellular endonuclease that cleaves the RNA strand of RNA:DNA duplexes;activation of this enzyme therefore results in cleavage of the RNAtarget, and thus can greatly enhance the efficiency of RNAi inhibition.Cleavage of the RNA target can be routinely demonstrated by gelelectrophoresis. In an embodiment, the chimeric oligonucleotide is alsomodified to enhance nuclease resistance. Cells contain a variety of exo-and endo-nucleases which can degrade nucleic acids. A number ofnucleotide and nucleoside modifications have been shown to make theoligonucleotide into which they are incorporated more resistant tonuclease digestion than the native oligodeoxynucleotide. Nucleaseresistance is routinely measured by incubating oligonucleotides withcellular extracts or isolated nuclease solutions and measuring theextent of intact oligonucleotide remaining over time, usually by gelelectrophoresis. Oligonucleotides which have been modified to enhancetheir nuclease resistance survive intact for a longer time thanunmodified oligonucleotides. A variety of oligonucleotide modificationshave been demonstrated to enhance or confer nuclease resistance.Oligonucleotides which contain at least one phosphorothioatemodification are presently more preferred. In some cases,oligonucleotide modifications which enhance target binding affinity arealso, independently, able to enhance nuclease resistance.

Specific examples of some preferred oligonucleotides envisioned for thisinvention include those comprising modified backbones, for example,phosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are oligonucleotideswith phosphorothioate backbones and those with heteroatom backbones,particularly CH2-NH—O—CH2, CH, —N(CH3)-O—CH2 [known as amethylene(methylimino) or MMI backbone], CH2-O—N(CH3)-CH2,CH2-N(CH3)-N(CH3)-CH2 and O—N(CH3)-CH2-CH2 backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH,). The amidebackbones disclosed by De Mesmaeker et al. (1995) Acc. Chem. Res.28:366-374 are also preferred. Also preferred are oligonucleotideshaving morpholino backbone structures (Summerton and Weller, U.S. Pat.No. 5,034,506). In other an embodiment, such as the peptide nucleic acid(PNA) backbone, the phosphodiester backbone of the oligonucleotide isreplaced with a polyamide backbone, the nucleotides being bound directlyor indirectly to the aza nitrogen atoms of the polyamide backbone.Oligonucleotides may also comprise one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH, SH, SCH3, F, OCN, OCH3, O(CH2)nCH3, O(CH2)nNH2 orO(CH2)nCH3 where n is from 1 to about 10; C1 to C10 lower alkyl,alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br, CN;CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2 CH3;ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleavinggroup; a reporter group; an intercalator; a group for improving thepharmacokinetic properties of an oligonucleotide; or a group forimproving the pharmacodynamic properties of an oligonucleotide and othersubstituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy [2′-O—CH2 CH2 OCH3, also known as2′-O-(2-methoxyethyl)]. Other preferred modifications include2′-methoxy(2′-O—CH3), 2′-propoxy(2′-OCH2CH2CH3) and 2′-fluoro (2′-F).Similar modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutyls inplace of the pentofuranosyl group.

Oligonucleotides may also include, additionally or alternatively,nucleobase (often referred to in the art simply as “base”) modificationsor substitutions. As used herein, “unmodified” or “natural” nucleotidesinclude adenine (A), guanine (G), thymine (T), cytosine (C) and uracil(U). Modified nucleotides include nucleotides found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′ deoxycytosine and often referred to in theart as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC andgentobiosyl HMC, as well as synthetic nucleotides, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and2,6-diaminopurine. A “universal” base known in the art, e.g., inosine,may be included. 5-Me-C substitutions have been shown to increasenucleic acid duplex stability by 0.6-1.2° C. and are presently preferredbase substitutions.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety, a cholesteryl moiety, analiphatic chain, e.g., dodecandiol or undecyl residues, a polyamine or apolyethylene glycol chain, or Adamantane acetic acid. Oligonucleotidescomprising lipophilic moieties, and methods for preparing sucholigonucleotides are known in the art, for example, U.S. Pat. Nos.5,138,045, 5,218,105 and 5,459,255.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even atwithin a single nucleoside within an oligonucleotide. The presentinvention also includes oligonucleotides which are chimericoligonucleotides as hereinbefore defined.

In another embodiment, the nucleic acid molecule of the presentinvention is conjugated with another moiety including but not limited toabasic nucleotides, polyether, polyamine, polyamides, peptides,carbohydrates, lipid, or polyhydrocarbon compounds. Those skilled in theart will recognize that these molecules can be linked to one or more ofany nucleotides comprising the nucleic acid molecule at severalpositions on the sugar, base or phosphate group.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of one of ordinary skill in the art. It is alsowell known to use similar techniques to prepare other oligonucleotidessuch as the phosphorothioates and alkylated derivatives. It is also wellknown to use similar techniques and commercially available modifiedamidites and controlled-pore glass (CPG) products such as biotin,fluorescein, acridine or psoralen-modified amidites and/or CPG(available from Glen Research, Sterling Va.) to synthesize fluorescentlylabeled, biotinylated or other modified oligonucleotides such ascholesterol-modified oligonucleotides.

In accordance with the invention, use of modifications such as the useof LNA monomers to enhance the potency, specificity and duration ofaction and broaden the routes of administration of oligonucleotidescomprised of current chemistries such as MOE, ANA, FANA, PS etc. Thiscan be achieved by substituting some of the monomers in the currentoligonucleotides by LNA monomers. The LNA modified oligonucleotide mayhave a size similar to the parent compound or may be larger orpreferably smaller. It is preferred that such LNA-modifiedoligonucleotides contain less than about 70%, more preferably less thanabout 60%, most preferably less than about 50% LNA monomers and thattheir sizes are between about 5 and 25 nucleotides, more preferablybetween about 12 and 20 nucleotides.

Preferred modified oligonucleotide backbones comprise, but not limitedto, phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates comprising 3′ alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates comprising 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus containing linkages comprise, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainallyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides comprise, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds comprise, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen, et al. (1991) Science 254, 1497-1500.

In an embodiment of the invention the oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH2-NH—O—CH2-, —CH2-N(CH3)-O—CH2-known asa methylene (methylimino) or MMI backbone, —CH2-O—N(CH3)-CH2-,—CH2N(CH3)-N(CH3)CH2- and —O—N(CH3)-CH2-CH2- wherein the nativephosphodiester backbone is represented as —O—P—O—CH2- of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C1 to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are 0 (CH2)nOmCH3, O(CH2)nOCH3,O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON(CH3)2 where n and mcan be from 1 to about 10. Other preferred oligonucleotides comprise oneof the following at the 2′ position: C to CO, (lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN,Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification comprises 2′-methoxyethoxy (2′-O—CH2CH2OCH3,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) i.e., an alkoxyalkoxygroup. A further preferred modification comprises2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH2-O—CH2-N(CH2)2.

Other preferred modifications comprise 2′-methoxy(2′-OCH3),2′-aminopropoxy(2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures comprise, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference.

Oligonucleotides may also comprise nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleotides comprise the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleotides comprise other synthetic andnatural nucleotides such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further, nucleotides comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience And Engineering’, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., ‘AngewandleChemie, International Edition’, 1991, 30, page 613, and those disclosedby Sanghvi, Y. S., Chapter 15, ‘Antisense Research and Applications’,pages 289-302, Crooke, S. T. and Lebleu, B. ea, CRC Press, 1993. Certainof these nucleotides are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These comprise5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, comprising 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Research andApplications’, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-Omethoxyethyl sugar modifications.

Representative United States patents that teach the preparation of theabove noted modified nucleotides as well as other modified nucleotidescomprise, but are not limited to, U.S. Pat. No. 3,687,808, as well asU.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941,each of which is herein incorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates, which enhance the activity, cellular distribution, orcellular uptake of the oligonucleotide.

Such moieties comprise but are not limited to, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, apolyamine or a polyethylene glycol chain, or Adamantane acetic acid, apalmityl moiety, or an octadecylamine or hexylamino-carbonyl-toxycholesterol moiety.

Representative United States patents that teach the preparation of sucholigonucleotides conjugates comprise, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

Drug Discovery:

The compounds of the present invention can also be applied in the areasof drug discovery and target validation. The present inventioncomprehends the use of the compounds and preferred target segmentsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between Alpha-L-Iduronidase (IDUA) polynucleotides and adisease state, phenotype, or condition. These methods include detectingor modulating IDUA polynucleotides comprising contacting a sample,tissue, cell, or organism with the compounds of the present invention,measuring the nucleic acid or protein level of IDUA polynucleotidesand/or a related phenotypic or chemical endpoint at some time aftertreatment, and optionally comparing the measured value to a non-treatedsample or sample treated with a further compound of the invention. Thesemethods can also be performed in parallel or in combination with otherexperiments to determine the function of unknown genes for the processof target validation or to determine the validity of a particular geneproduct as a target for treatment or prevention of a particular disease,condition, or phenotype.

Assessing Up-regulation or Inhibition of Gene Expression:

Transfer of an exogenous nucleic acid into a host cell or organism canbe assessed by directly detecting the presence of the nucleic acid inthe cell or organism. Such detection can be achieved by several methodswell known in the art. For example, the presence of the exogenousnucleic acid can be detected by Southern blot or by a polymerase chainreaction (PCR) technique using primers that specifically amplifynucleotide sequences associated with the nucleic acid. Expression of theexogenous nucleic acids can also be measured using conventional methodsincluding gene expression analysis. For instance, mRNA produced from anexogenous nucleic acid can be detected and quantified using a Northernblot and reverse transcription PCR (RT-PCR).

Expression of RNA from the exogenous nucleic acid can also be detectedby measuring an enzymatic activity or a reporter protein activity. Forexample, antisense modulatory activity can be measured indirectly as adecrease or increase in target nucleic acid expression as an indicationthat the exogenous nucleic acid is producing the effector RNA. Based onsequence conservation, primers can be designed and used to amplifycoding regions of the target genes. Initially, the most highly expressedcoding region from each gene can be used to build a model control gene,although any coding or non coding region can be used. Each control geneis assembled by inserting each coding region between a reporter codingregion and its poly(A) signal. These plasmids would produce an mRNA witha reporter gene in the upstream portion of the gene and a potential RNAitarget in the 3′ non-coding region. The effectiveness of individualantisense oligonucleotides would be assayed by modulation of thereporter gene. Reporter genes useful in the methods of the presentinvention include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP),cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase(Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivativesthereof. Multiple selectable markers are available that conferresistance to ampicillin, bleomycin, chloramphenicol, gentamycin,hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin,puromycin, and tetracycline. Methods to determine modulation of areporter gene are well known in the art, and include, but are notlimited to, fluorometric methods (e.g. fluorescence spectroscopy,Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy),antibiotic resistance determination.

IDUA protein and mRNA expression can be assayed using methods known tothose of skill in the art and described elsewhere herein. For example,immunoassays such as the ELISA can be used to measure protein levels.IDUA ELISA assay kits are available commercially, e.g., from R&D Systems(Minneapolis, Minn.).

In embodiments, IDUA expression (e.g., mRNA or protein) in a sample(e.g., cells or tissues in vivo or in vitro) treated using an antisenseoligonucleotide of the invention is evaluated by comparison with IDUAexpression in a control sample. For example, expression of the proteinor nucleic acid can be compared using methods known to those of skill inthe art with that in a mock-treated or untreated sample. Alternatively,comparison with a sample treated with a control antisenseoligonucleotide (e.g., one having an altered or different sequence) canbe made depending on the information desired. In another embodiment, adifference in the expression of the IDUA protein or nucleic acid in atreated vs. an untreated sample can be compared with the difference inexpression of a different nucleic acid (including any standard deemedappropriate by the researcher, e.g., a housekeeping gene) in a treatedsample vs. an untreated sample.

Observed differences can be expressed as desired, e.g., in the form of aratio or fraction, for use in a comparison with control. In embodiments,the level of IDUA mRNA or protein, in a sample treated with an antisenseoligonucleotide of the present invention, is increased or decreased byabout 1.25-fold to about 10-fold or more relative to an untreated sampleor a sample treated with a control nucleic acid. In embodiments, thelevel of IDUA mRNA or protein is increased or decreased by at leastabout 1.25-fold, at least about 1.3-fold, at least about 1.4-fold, atleast about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold,at least about 1.8-fold, at least about 2-fold, at least about 2.5-fold,at least about 3-fold, at least about 3.5-fold, at least about 4-fold,at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold,at least about 6-fold, at least about 6.5-fold, at least about 7-fold,at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold,at least about 9-fold, at least about 9.5-fold, or at least about10-fold or more.

Kits, Research Reagents, Diagnostics, and Therapeutics

The compounds of the present invention can be utilized for diagnostics,therapeutics, and prophylaxis, and as research reagents and componentsof kits. Furthermore, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes orto distinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics and in various biological systems, thecompounds of the present invention, either alone or in combination withother compounds or therapeutics, are useful as tools in differentialand/or combinatorial analyses to elucidate expression patterns of aportion or the entire complement of genes expressed within cells andtissues.

As used herein the term “biological system” or “system” is defined asany organism, cell, cell culture or tissue that expresses, or is madecompetent to express products of the Alpha-L-Iduronidase (IDUA) genes.These include, but are not limited to, humans, transgenic animals,cells, cell cultures, tissues, xenografts, transplants and combinationsthereof.

As one non limiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundsthat affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays, SAGE (serial analysis of gene expression),READS (restriction enzyme amplification of digested cDNAs), TOGA (totalgene expression analysis), protein arrays and proteomics, expressedsequence tag (EST) sequencing, subtractive RNA fingerprinting (SuRF),subtractive cloning, differential display (DD), comparative genomichybridization, FISH (fluorescent in situ hybridization) techniques andmass spectrometry methods.

The compounds of the invention are useful for research and diagnostics,because these compounds hybridize to nucleic acids encodingAlpha-L-Iduronidase (IDUA). For example, oligonucleotides that hybridizewith such efficiency and under such conditions as disclosed herein as tobe effective IDUA modulators are effective primers or probes underconditions favoring gene amplification or detection, respectively. Theseprimers and probes are useful in methods requiring the specificdetection of nucleic acid molecules encoding IDUA and in theamplification of said nucleic acid molecules for detection or for use infurther studies of IDUA. Hybridization of the antisenseoligonucleotides, particularly the primers and probes, of the inventionwith a nucleic acid encoding IDUA can be detected by means known in theart. Such means may include conjugation of an enzyme to theoligonucleotide, radiolabeling of the oligonucleotide, or any othersuitable detection means. Kits using such detection means for detectingthe level of IDUA in a sample may also be prepared.

The specificity and sensitivity of antisense are also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs have beensafely and effectively administered to humans and numerous clinicaltrials are presently underway. It is thus established that antisensecompounds can be useful therapeutic modalities that can be configured tobe useful in treatment regimes for the treatment of cells, tissues andanimals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression ofIDUA polynucleotides is treated by administering antisense compounds inaccordance with this invention. For example, in one non-limitingembodiment, the methods comprise the step of administering to the animalin need of treatment, a therapeutically effective amount of IDUAmodulator. The IDUA modulators of the present invention effectivelymodulate the activity of the IDUA or modulate the expression of the IDUAprotein. In one embodiment, the activity or expression of IDUA in ananimal is inhibited by about 10% as compared to a control. Preferably,the activity or expression of IDUA in an animal is inhibited by about30%. More preferably, the activity or expression of IDUA in an animal isinhibited by 50% or more. Thus, the oligomeric compounds modulateexpression of Alpha-L-Iduronidase (IDUA) mRNA by at least 10%, by atleast 50%, by at least 25%, by at least 30%, by at least 40%, by atleast 50%, by at least 60%, by at least 70%, by at least 75%, by atleast 80%, by at least 85%, by at least 90%, by at least 95%, by atleast 98%, by at least 99%, or by 100% as compared to a control.

In one embodiment, the activity or expression of Alpha-L-Iduronidase(IDUA) in an animal is increased by about 10% as compared to a control.Preferably, the activity or expression of IDUA in an animal is increasedby about 30%. More preferably, the activity or expression of IDUA in ananimal is increased by 50% or more. Thus, the oligomeric compoundsmodulate expression of IDUA mRNA by at least 10%, by at least 50%, by atleast 25%, by at least 30%, by at least 40%, by at least 50%, by atleast 60%, by at least 70%, by at least 75%, by at least 80%, by atleast 85%, by at least 90%, by at least 95%, by at least 98%, by atleast 99%, or by 100% as compared to a control.

For example, the increase or reduction of the expression ofAlpha-L-Iduronidase (IDUA) may be measured in serum, blood, adiposetissue, liver or any other body fluid, tissue or organ of the animal.Preferably, the cells contained within said fluids, tissues or organsbeing analyzed contain a nucleic acid molecule encoding IDUA peptidesand/or the IDUA protein itself.

The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

Conjugates

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typicalconjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application No. PCT/US92/09196, filed Oct. 23,1992, and U.S. Pat. No. 6,287,860, which are incorporated herein byreference. Conjugate moieties include, but are not limited to, lipidmoieties such as a cholesterol moiety, cholic acid, a thioether, e.g.,hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H phosphonate, a polyamine or apolyethylene glycol chain, or Adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic.

Representative United States patents that teach the preparation of sucholigonucleotides conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

Formulations

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,165; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

Although, the antisense oligonucleotides do not need to be administeredin the context of a vector in order to modulate a target expressionand/or function, embodiments of the invention relates to expressionvector constructs for the expression of antisense oligonucleotides,comprising promoters, hybrid promoter gene sequences and possess astrong constitutive promoter activity, or a promoter activity which canbe induced in the desired case.

In an embodiment, invention practice involves administering at least oneof the foregoing antisense oligonucleotides with a suitable nucleic aciddelivery system. In one embodiment, that system includes a non-viralvector operably linked to the polynucleotide. Examples of such nonviralvectors include the oligonucleotide alone (e.g. any one or more of SEQID NOS: 10 to 28) or in combination with a suitable protein,polysaccharide or lipid formulation.

Additionally suitable nucleic acid delivery systems include viralvector, typically sequence from at least one of an adenovirus,adenovirus-associated virus (AAV), helper-dependent adenovirus,retrovirus, or hemagglutinatin virus of Japan-liposome (HVJ) complex.Preferably, the viral vector comprises a strong eukaryotic promoteroperably linked to the polynucleotide e.g., a cytomegalovirus (CMV)promoter.

Additionally preferred vectors include viral vectors, fusion proteinsand chemical conjugates. Retroviral vectors include Moloney murineleukemia viruses and HIV-based viruses. One preferred HIV-based viralvector comprises at least two vectors wherein the gag and pol genes arefrom an HIV genome and the env gene is from another virus. DNA viralvectors are preferred. These vectors include pox vectors such asorthopox or avipox vectors, herpesvirus vectors such as a herpes simplexI virus (HSV) vector, Adenovirus Vectors and Adeno-associated VirusVectors.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, preferred examples of pharmaceutically acceptablesalts and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein by reference.

The present invention also includes pharmaceutical compositions andformulations that include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer, intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration.

For treating tissues in the central nervous system, administration canbe made by, e.g., injection or infusion into the cerebrospinal fluid.Administration of antisense RNA into cerebrospinal fluid is described,e.g., in U.S. Pat. App. Pub. No. 2007/0117772, “Methods for slowingfamilial ALS disease progression,” incorporated herein by reference inits entirety.

When it is intended that the antisense oligonucleotide of the presentinvention be administered to cells in the central nervous system,administration can be with one or more agents capable of promotingpenetration of the subject antisense oligonucleotide across theblood-brain barrier. Injection can be made, e.g., in the entorhinalcortex or hippocampus. Delivery of neurotrophic factors byadministration of an adenovirus vector to motor neurons in muscle tissueis described in, e.g., U.S. Pat. No. 6,632,427,“Adenoviral-vector-mediated gene transfer into medullary motor neurons,”incorporated herein by reference. Delivery of vectors directly to thebrain, e.g., the striatum, the thalamus, the hippocampus, or thesubstantia nigra, is known in the art and described, e.g., in U.S. Pat.No. 6,756,523, “Adenovirus vectors for the transfer of foreign genesinto cells of the central nervous system particularly in brain,”incorporated herein by reference. Administration can be rapid as byinjection or made over a period of time as by slow infusion oradministration of slow release formulations.

The subject antisense oligonucleotides can also be linked or conjugatedwith agents that provide desirable pharmaceutical or pharmacodynamicproperties. For example, the antisense oligonucleotide can be coupled toany substance, known in the art to promote penetration or transportacross the blood-brain barrier, such as an antibody to the transferrinreceptor, and administered by intravenous injection. The antisensecompound can be linked with a viral vector, for example, that makes theantisense compound more effective and/or increases the transport of theantisense compound across the blood-brain barrier. Osmotic blood brainbarrier disruption can also be accomplished by, e.g., infusion of sugarsincluding, but not limited to, meso erythritol, xylitol, D(+) galactose,D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(−) fructose, D(−)mannitol, D(+) glucose, D(+) arabinose, D(−) arabinose, cellobiose, D(+)maltose, D(+) raffmose, L(+) rhammose, D(+) melibiose, D(−) ribose,adonitol, D(+) arabitol, L(−) arabitol, D(+) fucose, L(−) fucose, D(−)lyxose, L(+) lyxose, and L(−) lyxose, or amino acids including, but notlimited to, glutamine, lysine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glycine, histidine, leucine, methionine,phenylalanine, proline, serine, threonine, tyrosine, valine, andtaurine. Methods and materials for enhancing blood brain barrierpenetration are described, e.g., in U.S. Pat. No. 4,866,042, “Method forthe delivery of genetic material across the blood brain barrier,”6,294,520, “Material for passage through the blood-brain barrier,” and6,936,589, “Parenteral delivery systems,” all incorporated herein byreference in their entirety.

The subject antisense compounds may be admixed, encapsulated, conjugatedor otherwise associated with other molecules, molecule structures ormixtures of compounds, for example, liposomes, receptor-targetedmolecules, oral, rectal, topical or other formulations, for assisting inuptake, distribution and/or absorption. For example, cationic lipids maybe included in the formulation to facilitate oligonucleotide uptake. Onesuch composition shown to facilitate uptake is LIPOFECTIN (availablefrom GIBCO-BRL, Bethesda, Md.).

Oligonucleotides with at least one 2′-O-methoxyethyl modification arebelieved to be particularly useful for oral administration.Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances that increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogeneous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug that may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes that are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids. When incorporated into liposomes, these specialized lipidsresult in liposomes with enhanced circulation lifetimes relative toliposome slacking such specialized lipids. Examples of stericallystabilized liposomes are those in which part of the vesicle-forminglipid portion of the liposome comprises one or more glycolipids or isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. Liposomes and their uses are furtherdescribed in U.S. Pat. No. 6,287,860.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein by reference.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating nonsurfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein by reference.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoyl-phosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoyl-phosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein by reference. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein by reference.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents that function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited tocancer chemotherapeutic drugs such as daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bischloroethyl-nitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclo-phosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. For example, the first targetmay be a particular antisense sequence of Alpha-L-Iduronidase (IDUA),and the second target may be a region from another nucleotide sequence.Alternatively, compositions of the invention may contain two or moreantisense compounds targeted to different regions of the sameAlpha-L-Iduronidase (IDUA) nucleic acid target. Numerous examples ofantisense compounds are illustrated herein and others may be selectedfrom among suitable compounds known in the art. Two or more combinedcompounds may be used together or sequentially.

Dosing:

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC50s found to be effective invitro and in vivo animal models. In general, dosage is from 0.01 μg toabout 10 mg per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to about 10 mgper kg of body weight, once or more daily, to once every 2-20 years.

In embodiments, a patient is treated with a dosage of drug that is atleast about 1, at least about 2, at least about 3, at least about 4, atleast about 5, at least about 6, at least about 7, at least about 8, atleast about 9, at least about 10, at least about 15, at least about 20,at least about 25, at least about 30, at least about 35, at least about40, at least about 45, at least about 50, at least about 60, at leastabout 70, at least about 80, at least about 90, or at least about 10mg/kg body weight. Certain injected dosages of antisenseoligonucleotides are described, e.g., in U.S. Pat. No. 7,563,884,“Antisense modulation of PTP1B expression,” incorporated herein byreference in its entirety. MPS-1 (i.e. mucopolysaccharidosis type 1) isa rare lysosomal storage disease. This disease has three groups ofpatients with distinct symptoms based on the severity of the disease(Hurler syndrome, Hurler-Scheie syndrome, Scheie syndrome). In studiesto determine and support a method of determining and selecting the mostpreferred oligonucleotide for any individual patient or group ofpatients having the disease, the following general protocol may beperformed. This method may of course use any cells or tissues typicallyhaving IDUA polynucleotides and expression products derived therefrom. Apatient population may be selected using the following criteria 1, 2;then after acceptance, steps 3, 4, 5 and 6 are performed: (1) Thepatients with show MPS-1 (i.e. mucopolysaccharidosis type 1) due to adeficiency in IDUA activity. (2) These patients will be defined frommedical exam as having a Hurler syndrome or a Hurler-Scheie syndrome ora Scheie syndrome. (3) After patient/guardian consent, a skin biopsywill be taken from the patient; the patient will also be checked for anyother diseases (for example infectious diseases) that would requirespecial precautions when handling biological samples from the patient(4) After full documentation on the patient conditions, the skin bioposywill processed to expand skin fibroblasts in vitro. (5) The skinfibroblasts will be dosed with different concentrations of oligos anddifferent oligos; the oligos are a selected set of oligos complementaryto the human IDUA natural antisense that would have been previouslycharacterized as up-regulating the IDUA (mRNA, protein and activity).(6) The percentage increase in IDUA activity is measured from the skinfibroblast cell culture supernatant. NCBI (The National Center forBiotechnology Information) characterizes IDUA activity in differentpatients (or control) subsets as follows: a patient with two wild typeIDUA alleles (IDUA gene from each parent is wild type, meaning has nomutation) the activity of IDUA is 83-121%; Patients with one strongmutation in IDUA (heterozygotes), the IDUA activity is 19 to 60%;Patients with two very strong mutations in IDUA, the total IDUA activityis 0-3%. Heterozygotes are only carriers of the disease and do not showsymptoms of the disease. The oligos increasing the IDUA activity to morethan about 10% of the total activity seen in normal cells could beconsidered active drug candidates. Preferrably, the percentage increasewill be above about 20%. The oligonucleotide with the highest percentageincrease in IDUA upregulation is selected as the drug candidate for theindividual patient from which the fibroblast measurement was made. Theoligonucleotide may also be useful in a subset of patients having thesame disease condition or to treat the disease in all of the patientshaving such disease.

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. Numerous changes to the disclosedembodiments can be made in accordance with the disclosure herein withoutdeparting from the spirit or scope of the invention. Thus, the breadthand scope of the present invention should not be limited by any of theabove described embodiments.

All documents mentioned herein are incorporated herein by reference. Allpublications and patent documents cited in this application areincorporated by reference for all purposes to the same extent as if eachindividual publication or patent document were so individually denoted.By their citation of various references in this document, Applicants donot admit any particular reference is “prior art” to their invention.Embodiments of inventive compositions and methods are illustrated in thefollowing examples.

EXAMPLES

The following non-limiting Examples serve to illustrate selectedembodiments of the invention. It will be appreciated that variations inproportions and alternatives in elements of the components shown will beapparent to those skilled in the art and are within the scope ofembodiments of the present invention.

Example 1 Design of Antisense Oligonucleotides Specific for a NucleicAcid Molecule Antisense to an Alpha-L Iduronidase (IDUA) and/or a SenseStrand of IDUA Polynucleotide

As indicated above the term “oligonucleotide specific for” or“oligonucleotide targets” refers to an oligonucleotide having a sequence(i) capable of forming a stable complex with a portion of the targetedgene, or (ii) capable of forming a stable duplex with a portion of anmRNA transcript of the targeted gene.

Selection of appropriate oligonucleotides is facilitated by usingcomputer programs (e.g. IDT AntiSense Design, IDT OligoAnalyzer) thatautomatically identify in each given sequence subsequences of 19-25nucleotides that will form hybrids with a target polynucleotide sequencewith a desired melting temperature (usually 50-60° C.) and will not formself-dimers or other complex secondary structures.

Selection of appropriate oligonucleotides is further facilitated byusing computer programs that automatically align nucleic acid sequencesand indicate regions of identity or homology. Such programs are used tocompare nucleic acid sequences obtained, for example, by searchingdatabases such as GenBank or by sequencing PCR products. Comparison ofnucleic acid sequences from a range of genes and intergenic regions of agiven genome allows the selection of nucleic acid sequences that displayan appropriate degree of specificity to the gene of interest. Theseprocedures allow the selection of oligonucleotides that exhibit a highdegree of complementarity to target nucleic acid sequences and a lowerdegree of complementarity to other nucleic acid sequences in a givengenome. One skilled in the art will realize that there is considerablelatitude in selecting appropriate regions of genes for use in thepresent invention.

An antisense compound is “specifically hybridizable” when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a modulation of function and/oractivity, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo assays ortherapeutic treatment, and under conditions in which assays areperformed in the case of in vitro assays.

The hybridization properties of the oligonucleotides described hereincan be determined by one or more in vitro assays as known in the art.For example, the properties of the oligonucleotides described herein canbe obtained by determination of binding strength between the targetnatural antisense and a potential drug molecules using melting curveassay.

The binding strength between the target natural antisense and apotential drug molecule (Molecule) can be estimated using any of theestablished methods of measuring the strength of intermolecularinteractions, for example, a melting curve assay.

Melting curve assay determines the temperature at which a rapidtransition from double-stranded to single-stranded conformation occursfor the natural antisense/Molecule complex. This temperature is widelyaccepted as a reliable measure of the interaction strength between thetwo molecules.

A melting curve assay can be performed using a cDNA copy of the actualnatural antisense RNA molecule or a synthetic DNA or RNA nucleotidecorresponding to the binding site of the Molecule. Multiple kitscontaining all necessary reagents to perform this assay are available(e.g. Applied Biosystems Inc. MeltDoctor kit). These kits include asuitable buffer solution containing one of the double strand DNA (dsDNA)binding dyes (such as ABI FIRM dyes, SYBR Green, SYTO, etc.). Theproperties of the dsDNA dyes are such that they emit almost nofluorescence in free form, but are highly fluorescent when bound todsDNA.

To perform the assay the cDNA or a corresponding oligonucleotide aremixed with Molecule in concentrations defined by the particularmanufacturer's protocols. The mixture is heated to 95° C. to dissociateall pre-formed dsDNA complexes, then slowly cooled to room temperatureor other lower temperature defined by the kit manufacturer to allow theDNA molecules to anneal. The newly formed complexes are then slowlyheated to 95° C. with simultaneous continuous collection of data on theamount of fluorescence that is produced by the reaction. Thefluorescence intensity is inversely proportional to the amounts of dsDNApresent in the reaction. The data can be collected using a real time PCRinstrument compatible with the kit (e.g. ABI's StepOne Plus Real TimePCR System or lightTyper instrument, Roche Diagnostics, Lewes, UK).

Melting peaks are constructed by plotting the negative derivative offluorescence with respect to temperature (−d(Fluorescence)/dT) on they-axis) against temperature (x-axis) using appropriate software (forexample lightTyper (Roche) or SDS Dissociation Curve, ABI). The data isanalyzed to identify the temperature of the rapid transition from dsDNAcomplex to single strand molecules. This temperature is called Tm and isdirectly proportional to the strength of interaction between the twomolecules. Typically, Tm will exceed 40° C.

Example 2 Modulation of IDUA Polynucleotides

All antisense oligonucleotides used in Example 2 were designed asdescribed in Example 1. The manufacturer (IDT Inc. of Coralville, Iowa)was instructed to manufacture the designed phosphothioate bondoligonucleotides and provided the designed phosphothioate analogs shownin Table 1. The asterisk designation between nucleotides indicates thepresence of phosphothioate bond. The oligonucleotides required for theexperiment in Example 2 can be synthesized using any appropriate stateof the art method, for example the method used by IDT: on solid support,such as a 5 micron controlled pore glass bead (CPG), usingphosphoramidite monomers (normal nucleotides with all active groupsprotected with protection groups, e.g. trityl group on sugar, benzoyl onA and C and N-2-isobutyryl on G). Protection groups prevent the unwantedreactions during oligonucleotide synthesis. Protection groups areremoved at the end of the synthesis process. The initial nucleotide islinked to the solid support through the 3′ carbon and the synthesisproceeds in the 3′ to 5′ direction. The addition of a new base to agrowing oligonucleotide chain takes place in four steps: 1) theprotection group is removed from the 5′ oxygen of the immobilizednucleotide using trichloroacetic acid; 2) the immobilized and thenext-in-sequence nucleotides are coupled together using tetrazole; thereaction proceeds through a tetrazolyl phosphoramidite intermediate; 3)the unreacted free nucleotides and reaction byproducts are washed awayand the unreacted immobilized oligonucleotides are capped to preventtheir participation in the next round of synthesis; capping is achievedby acetylating the free 5′ hydroxyl using acetic anhydride and N-methylimidazole; 4) to stabilize the bond between the nucleotides thephosphorus is oxidized using iodine and water, if a phosphodiester bondis to be produced, or Beaucage reagent(3H-1,2-benzodithiol-3-one-1,1-dioxide), if a phosphothioate bond isdesired. By alternating the two oxidizing agents, a chimeric backbonecan be constructed. The four step cycle described above is repeated forevery nucleotide in the sequence. When the complete sequence issynthesized, the oligonucleotide is cleaved from the solid support anddeprotected using ammonium hydroxide at high temperature. Protectiongroups are washed away by desalting and the remaining oligonucleotidesare lyophilized.

Treatment of HepG2 Cells with Antisense Oligonucleotides

To perform the experiment designed in Example 2, HepG2 cells from ATCC(cat#HB-8065) were grown in growth media (MEM/EBSS (Hyclone cat#SH30024, or Mediatech cat #MT-10-010-CV)+10% FBS (Mediatechcat#MT35-011-CV)+penicillin/streptomycin (Mediatech cat#MT30-002-CI)) at37° C. and 5% CO2. One day before the experiment the cells were replatedat the density of 0.5×104/ml into 6 well plates and incubated at 37° C.and 5% CO2 overnight. On the day of the experiment the media in the 6well plates was changed to fresh growth media.

Oligonucleotides shipped by the manufacturer in lyophilized form werediluted to the concentration of 20 in deionized RNAse/DNAse-free water.Two μl of this solution was incubated with 400 μl of OptiMEM media(Gibco cat#31985-070) and 4 μl of Lipofectamine 2000 (Invitrogencat#11668019) at room temperature for 20 min, then applied dropwise toone well of the 6 well plate with HcpG2 cells. Similar mixture including2 μl of water instead of the oligonucleotide solution was used for themock-transfected controls. After 3-18 h of incubation at 37° C. and 5%CO2 the media was changed to fresh growth media. 48 h after addition ofantisense oligonucleotides the media was removed and RNA was extractedfrom the cells using SV Total RNA Isolation System from Promega (cat#Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat#74181)following the manufacturers' instructions. 600 ng of extracted RNA wasadded to the reverse transcription reaction performed using Verso cDNAkit from Thermo Scientific (cat#AB1453B) or High Capacity cDNA ReverseTranscription Kit (cat#4368813) as described in the manufacturer'sprotocol. The cDNA from this reverse transcription reaction was used tomonitor gene expression by real time PCR using ABI Taqman GeneExpression Mix (cat#4369510) and primers/probes designed by ABI (AppliedBiosystems Taqman Gene Expression Assay: Hs00164940_ml (IDUA) by AppliedBiosystems Inc., Foster City Calif.). The following PCR cycle was used:50° C. for 2 min, 95° C. for 10 min, 40 cycles of (95° C. for 15seconds, 60° C. for 1 min) using StepOne Plus Real Time PCR Machine(Applied Biosystems). Fold change in gene expression after treatmentwith antisense oligonucleotides was calculated based on the differencein 18S-normalized dCt values between treated and mock-transfectedsamples.

Results:

Real Time PCR results show that levels of IDUA mRNA in HepG2 cells aresignificantly increased 48 h after treatment with the antisenseoligonucleotides to human IDUA antisense Hs.656285 with the oligosCUR-1820 and CUR-1821 (FIG. 1), with the oligos CUR-1978, CUR-1984,CUR-1985, CUR-1987 and CUR1988 (FIG. 2), and with the oligos CUR-1974and CUR-1986 (FIG. 3).

Treatment of SK-N-AS Cells with Antisense Oligonucleotides

In this example SK-N-AS antisense oligonucleotides of differentchemistries targeting IDUA-specific natural antisense transcript werescreened in human neuroblastoma SK-N-AS cell line at a finalconcentration of 20 nM.

Materials and Methods:

SK-N-AS cell line. SK-N-AS human neuroblastoma cells from ATCC(cat#CRL-2137) were grown in Growth Media (DMEM (Mediatechcat#10-013-CV)+10% FBS (Mediatechcat#MT35-011-CV)+penicillin/streptomycin (Mediatechcat#MT30-002-CI)+Non-Essential Amino Acids (NEAA) (HyClone SH30238.01))at 37° C. and 5% CO2. The cells were treated with antisenseoligonucleotides using one of the following methods. For the Next DayMethod, one day before the experiment the cells were replated at thedensity of approximately 3×105/well into 6 well plates in Growth Mediaand incubated at 37° C. and 5% CO₂ overnight. Next day, the media in the6 well plates was changed to fresh Growth Media (1.5 ml/well) and thecells were dosed with antisense oligonucleotides. All antisenseoligonucleotides were manufactured by IDT Inc. (Coralville, Iowa) orExiqon (Vedbaek, Denmark). The sequences for all oligonucleotides arelisted in Table 1. Stock solutions of oligonucleotides were diluted tothe concentration of 20 μM in DNAse/RNAse-free sterile water. To doseone well, 1 μl of this solution was incubated with 200 μl of Opti-MEMmedia (Gibco cat#31985-070) and 2 μl of Lipofectamine 2000 (Invitrogencat#11668019) at room temperature for 20 min and applied dropwise to onewell of a 24 well plate with cells. Similar mixture including 1 μl ofwater instead of the oligonucleotide solution was used for themock-transfected controls. After about 18 h of incubation at 37° C. and5% CO₂ the media was changed to fresh Growth Media. Forty eight hoursafter addition of antisense oligonucleotides the media was removed andRNA was extracted from the cells using SV Total RNA Isolation Systemfrom Promega (cat #Z3105) following the manufacturers' instructions. Sixhundred nanograms of purified total RNA was added to the reversetranscription reaction performed using SuperScript VILO cDNA SynthesisKit from Invitrogen (cat#11754-250) as described in the manufacturer'sprotocol. The cDNA from this reverse transcription reaction was used tomonitor gene expression by real time PCR using ABI Taqman GeneExpression Mix (cat#4369510) and primers/probes designed by ABI (assaysHs00164940_ml). Results obtained using all three assays were verysimilar. The following PCR cycle was used: 50° C. for 2 min, 95° C. for10 min, 40 cycles of (95° C. for 15 seconds, 60° C. for 1 min) usingStepOne Plus Real Time PCR system (Applied Biosystems). The assay for18S was manufactured by ABI (cat#4319413E). Fold change in geneexpression after treatment with antisense oligonucleotides wascalculated based on the difference in 18S-normalized dCt values betweentreated and mock-transfected samples.

Results:

Real Time PCR results show that levels of IDUA mRNA in SK-N-AS cellsshow a trend for increased at 48 h after treatment with the antisenseoligonucleotides CUR-1976, CUR1978 and CUR-1987 to human IDUA naturalantisense Hs.656285 (FIG. 4).

Example 3 Extension of the Dog IDUA Potential Natural Antisense Sequence

The purpose of this experiment is to extend the known sequence of thedog IDUA natural antisense DN876121 by sequencing all its sequence. Theoriginal DN876121 RNA transcript was obtained from dog eye minus lensand cornea tissue. A directionally cloned cDNA library was prepared in apCMVSport6 vector (Invitrogen) at Bioserve Biotechnology by Laurel Md.This work was done by April 2005. The DN876121 clone is currentlyavailable at Open Biosystems (Open Biosystems Products, Huntsville,Ala.). In April 2005, the DN876121 clone was not sequenced completely.OPKO-CURNA obtained the DN876121 clone and sequenced the full insert. Toachieve this, a bacterial clone containing a plasmid with the DN876121insert was acquired from Open Biosystems and plated in a Luria Bertani(LB)-agar plate with ampicillin to isolate individual colonies. Thencolonies were amplified in 5 ml of LB broth. The plasmid containing theDN876121 insert was then isolated from these bacteria and sent forsequencing to Davis Sequencing (Davis, Calif.).

Material and Methods: Isolation and Sequencing of the Plasmid Containingthe cDNA for the Dog IDUA Potential Natural Antisense DN876121

Suspension of frozen bacteria containing the DN876121 plasmid waspurchased from Open Biosystems (Open Biosystems Products, cat#NAE04B03),diluted 1:10, 1:100, 1:1000, 1:10000, 1:100000 times, then plated onLuria Bertani (LB) (BD, cat#244520)-agar plate (Falcon, cat#351005) with100 μg/ml of ampicillin (Calbiochem, cat#171254). After 15 h, 20individual colonies of bacteria were isolated from the plate with the1:100000 dilution and grown separately in 5 ml of LB broth (FisherScientific, cat#BP1426-2) for 15 h-24 h. At this time, the bacteria werepelleted and the plasmid (containing the cDNA from the DN876121 RNAtranscript) was isolated using the PureYield™ Plasmid Miniprep Systemkit from Promega (Promega, cat#A1222) following the manufacturer'sprotocol. The isolated DNA was diluted to 200 ng/ml and 12 μl of plasmidfrom each colony was sent for sequencing to Davis sequencing (Davis,Calif.).

Results:

The sequences obtained from Davis sequencing showed a substantialextension of the known DN876121 sequence shown in FIG. 5.

Conclusion:

The successful extension of the known DN876121 sequence by 578nucleotides served as a basis to design antisense oligonucleotidesagainst the dog IDUA potential natural antisense transcriptDN876121-extended (SEQ ID NO:8).

Example 4 IDUA Activity

The purpose of this experiment is to rank compounds according to theirability to upregulate the IDUA activity in different cells using theenzymatic activity of IDUA. This method could be used to rankoligonucleotide complementary to the IDUA natural antisense known toup-regulate the IDUA mRNA (and IDUA protein) for their capacity toincrease the IDUA activity. This protocol in combination with thepatient fibroblast cell expansion protocol could allow to screen invitro for the correct oligonucleotide complementary to the IDUA naturalantisense able to increase the activity of IDUA before offering thisoligonucleotide as a treatment to a patient.

Materials and Methods:

Cells will be treated with oligonucleotides complementary to the IDUAnatural antisense at 0 to 80 nM using lipofectamine TM 2000. After 24 h,the medium will be discarded and fresh medium will be added for 24 h upto 72 h. At that time, the medium will be stored and checked for IDUAactivity. The IDUA activity will be measured using as controlrecombinant human IDUA (from R&D systems Inc. Minneapolis Minn.) serialdiluted (maximum concentration at 0.2 microg/mL) in assay buffer (50 mMNaOAc, 150 mMNaCl, 0.02% Brij-35 (w/v) pH3.5). An equal volume ofrecombinant human IDUA in assay buffer with IDUA susbtrate(4-methylumberlliferyl-alpha-L-iduronide) from Glycosynth (Warrington,UK) at 200 microM in assay buffer will be mixed in a 96 well plate (100microLeach reaction solution). Incubate for 10 min at room temperature.Dilute the mixtures for 0.005 microg/mL maximum recombinant human IDUA(and 5 microM of substrate) in developing buffer (0.1M Tris pH9.0). Load100 microL of the diluted reactions into a fluorescence assay plate. Thesolution is read at 365 nm and 445 nm. The specific activity will becalculated (pmoles/min/microg) as follow:

${{IDUA}\mspace{14mu}{activity}} = \frac{\begin{matrix}{{Adjusted}\mspace{14mu}{for}\mspace{14mu}{substrate}\mspace{14mu}{blank}\mspace{14mu}{{Fluorescence}({RFU})} \times} \\{{Conversion}\mspace{14mu}{{factor}( {{pmole}/{RFU}} )}}\end{matrix}}{{Incubation}\mspace{14mu}{{time}( \min )} \times {amount}\mspace{14mu}{of}\mspace{14mu}{{enzyme}({microg})}}$The IDUA activity will be measured in cell supernatant by adding cellsupernatant from cells treated with different amounts of differentoligonucleotides complementary of the IDUA natural antisense transcriptinstead of recombinant human IDUA in this protocol.

Example 5 IDUA Protein ELISA

The purpose of this experiment is to rank compounds according to theirability to upregulate the IDUA protein expression in different cellsusing a technique called enzyme-linked immunosorbent assay (ELISA).

Materials and Methods:

Amounts of IDUA protein produced by the cells will be quantified byELISA. To achieve this, the cells will be grown in 24-well plates usingappropriate growth conditions. Forty eight hours after addition of smallcompounds, the media will be removed and the cells will be washed 3times with Dulbecco's phosphate-buffered saline without calcium andmagnesium (PBS) (Mediatech cat#21-031-CV). Then PBS will be discardedand the cells will be fixed in the 24 well plate using 100 μl of 100%methanol for 15 min at −20° C. After removing the methanol and washingwith PBS, the cells will be incubated with 3% hydrogen peroxide (FisherChemical cat#H325-100) for 5 min at 21° C. The cells will be washedthree times for 5 min with PBS, then incubated with 100 μl of bovineserum albumin (BSA) (Sigma cat#A-9647) at 0.1% in PBS for 30 min at 21°C. The cells will be washed three times for 5 min with PBS thenincubated with 300 μl of avidin solution (Vector Laboratoriescat#SP-2001) for 30 min at 21° C. The cells will be briefly rinsed threetimes with PBS then incubated with biotin solution (Vector Laboratoriescat#SP-2001) for 30 min at 21° C. The cells will be washed three timeswith PBS and then incubated overnight at 4° C. with 100 μl per well ofrabbit antibody raised against a region within internal sequence aminoacids 244-274 of Human IDUA (Abeam cat#ab103949) in PBS/BSA 0.1%. Afterequilibrating the plate for 5 min at 21° C., the cells will be washedthree times for 5 min each with PBS then incubated with goat anti-rabbitantibody diluted 1:200 in PBS/BSA 0.1% for 30 min at 21° C. The cellswill be washed three times for 5 min with PBS and then incubated with300 μl of Vectastain Elite ABC reagent A+B solution (Vector Laboratoriescat#PK-6101) for 30 min; the Vectastain Elite ABC reagent A+B solutionwill be prepared at 21° C. 30 min before incubation with the cells byadding and mixing successively 2 drops of reagent A to 5 ml of PBS andthen 2 drops of reagent B. The cells will be washed 3 times for 5 minwith PBS at 21° C. and then incubated with tetramethylbenzidine (TMB)peroxidase substrate solution (Thermo Scientific cat#N301). After thesupernatant turns blue, it will be transferred to a new 96 well ELISAplate (Greiner bio one cat #65121) and 1 M sulfuric acid will be added.The absorbance will be read at 450 nm using a Multiskan Spectrumspectrophotometer (Thermo Scientific). The background signal, read inthe wells stained with a rabbit anti-mouse IgG as primary antibody(Abeam cat#ab6709) will be subtracted from all IDUA and actin readings.Rabbit anti-actin antibody from Abeam (cat#ab1801) will be used. TheIDUA signal will be normalized to actin signal for each condition andnormalized values for each experimental variant will be compared.

Example 6 IDUA Immune-Histochemistry

The purpose of this experiment is to rank compounds according to theirability to upregulate the IDUA protein expression in different cellsusing a technique called immunohistochemistry.

Materials and Methods:

IDUA protein will be detected inside cells by immunohistochemistry. Toachieve this, the cells will be grown in 24-well plates usingappropriate growth conditions. Forty eight hours after addition of smallcompounds, the media will be removed and the cells will be washed 3times with Dulbecco's phosphate-buffered saline without calcium andmagnesium (PBS) (Mcdiatcch cat#21-031-CV). Then PBS will be discardedand the cells will be fixed in the 24 well plate using 300 μl of 100%methanol for 15 min at −20° C. After removing the methanol and washingwith PBS, the cells will be incubated with 3% hydrogen peroxide (FisherChemical cat#H325-100) for 5 min at 21° C. The cells will be washedthree times for 5 min with PBS, then incubated with 300 μl of bovineserum albumin (BSA) (Sigma cat#A-9647) at 0.1% in PBS for 30 min at 21°C. The cells will be washed three times for 5 min with PBS thenincubated with 300 μl of avidin solution (Vector Laboratoriescat#SP-2001) for 30 min at 21° C. The cells will be briefly rinsed threetimes with PBS then incubated with biotin solution (Vector Laboratoriescat#SP-2001) for 30 min at 21° C. The cells will be washed three timeswith PBS and then incubated overnight at 4° C. with 300 μl per well ofrabbit antibody raised against a region within internal sequence aminoacids 244-274 of Human IDUA (Abeam cat#ab103949) in PBS/BSA 0.1%. Afterequilibrating the plate for 5 min at 21° C., the cells will be washedthree times 5 min each with PBS then incubated with goat anti-rabbitantibody diluted 1:200 in PBS/BSA 0.1% for 30 min at 21° C. The cellswill be washed three times for 5 min with PBS and then incubated with300 μl of Vectastain Elite ABC reagent A+B solution (Vector Laboratoriescat#PK-6101) for 30 min; the Vectastain Elite ABC reagent A+B solutionwill be prepared at 21° C. 30 min before incubation with the cells byadding and mixing successively 2 drops of reagent A to 5 ml of PBS andthen 2 drops of reagent B. The cells will be washed 3 times for 5 mineach with PBS at 21° C. and then incubated with Diaminobenzidine (DAB)peroxidase substrate solution (Vector Laboratories cat#SK-4105) untilcells are stained; the DAB peroxidase substrate solution will bereconstituted before being added to the cells by mixing 1 ml ofImmPACT™DAB Diluent with 30 μl of ImmPACT™ DAB Chromogen concentrate. Atthis time, the cells will be briefly washed three times with PBS and 300μl of PBS will be left in each well. The staining of the cells will beanalyzed directly inside the wells of the 24-well plate using aninverted Nikon Eclipse TS100 microscope equipped with a Nikon DS-Rilcamera coupled with Nikon Digital-Sight equipment on the screen of aDell Latitude D630 laptop. Photos of individual wells will be made usingthe software provided with the Nikon camera, the MS-Elements D 3.0.

Example 7 Patient Fibroblasts

The purpose of this experiment is to identify the light oligonucleotideknown to up-regulate IDUA mRNA in the right patient population. The IDUAmutation is also present in the patient skin fibroblast cells. By dosingsuch cells in vitro, it will be possible to identify whicholigonucleotide complementary to the IDUA natural antisense could helppatient benefit from this innovative treatment.

Materials and Methods:

A skin biopsy will be performed according to the FDA regulations andwith patient consent in order to expand the patient skin fibroblasts incell culture for in vitro testing of the oligonucleotides complementaryto the IDUA natural antisense. This biopsy will be treated withcollagenase in order to dissociate the skin cells and this cellsuspension will be plated in wells of 6-well plates in 2 ml ofDulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (Invitrogencat#10565) with 20% Fetal Bovine Serum (from GIBO/Invitrogen Cat.#35-011CV). When the cells reach 70% confluence, they will be splited at1:4 in 2 ml of Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12(Invitrogen cat#10565) with 20% Fetal Bovine Serum (from GIBO/InvitrogenCat #35-011CV). These cells will be dosed with oligonucleotidescomplementary to the IDUA natural antisense following the same protocolas describe previously to check for IDUA mRNA up-regulation. The totalcell RNA will be checked for up-regulation of the IDUA mRNA after dosingwith oligonucleotides complementary to the IDUA natural antisensetranscript. The supernatant of these cells will be checked forup-regulation of the IDUA activity after dosing with oligonucleotidescomplementary to the IDUA natural antisense transcript.

Although the invention has been illustrated and described with respectto one or more implementations, equivalent alterations and modificationswill occur to others skilled in the art upon the reading andunderstanding of this specification and the annexed drawings. Inaddition, while a particular feature of the invention may have beendisclosed with respect to only one of several implementations, suchfeature may be combined with one or more other features of the otherimplementations as may be desired and advantageous for any given orparticular application.

The Abstract of the disclosure will allow the reader to quicklyascertain the nature of the technical disclosure. It is submitted withthe understanding that it will not be used to interpret or limit thescope or meaning of the following claims.

What is claimed is:
 1. A method of upregulating a function of and/or theexpression of an Alpha-L-Iduronidase (IDUA) polynucleotide having SEQ IDNO: 1 in a biological system comprising: contacting said biologicalsystem with at least one single stranded antisense oligonucleotide 12 to30 nucleotides in length wherein said at least one oligonucleotide hasat least sequence identity to a reverse complement of a polynucleotidecomprising 12 to 30 consecutive nucleotides within the natural antisensetranscript nucleotides 1 to 2695 of SEQ ID NO: 2 or 1 to 2082 of SEQ IDNO: 3 or 1 to 2739 of SEQ ID NO: 9; thereby upregulating a function ofand/or the expression of the Alpha-L-Iduronidase (IDUA) polynucleotide.2. A method of upregulating a function of and/or the expression of anAlpha-L-Iduronidase (IDUA) polynucleotide having SEQ ID NO: 1 in abiological system comprising: contacting said system with at least onesingle stranded antisense oligonucleotide of 10 to 30 nucleotides inlength that targets and specifically hybridizes to a region of a naturalantisense polynucleotide of the Alpha-L-Iduronidase (IDUA)polynucleotide selected from the group consisting of SEQ ID NOS: 2 or 9;thereby upregulating a function of and/or the expression of theAlpha-L-Iduronidase (IDUA) polynucleotide.
 3. The method of claim 2,wherein a function of and/or the expression of the Alpha-L-Iduronidase(IDUA) is increased in vivo or in vitro with respect to a control. 4.The method according to claim 3 wherein the increase in expressionrelative to control is greater than about fifteen (15) percent.
 5. Themethod according to claim 3 wherein the increase in expression relativeto control is greater than about nineteen (19) percent.
 6. The methodaccording to claim 3 wherein the increase in expression relative tocontrol is greater than about twenty-five (25) percent.
 7. The method ofclaim 2, wherein the at least one antisense oligonucleotide targets anatural antisense sequence of an Alpha-L-Iduronidase (IDUA)polynucleotide selected from SEQ ID NO:
 2. 8. The method of claim 2,wherein the at least one antisense oligonucleotide targets a naturalantisense polynucleotide antisense to coding nucleic acid sequences ofan Alpha-L-Iduronidase (IDUA) polynucleotide.
 9. The method of claim 2,wherein the at least one antisense oligonucleotide targets a naturalantisense polynucleotide having overlapping sequences with anAlpha-L-Iduronidase (IDUA) polynucleotide.
 10. The method of claim 2,wherein the at least one antisense oligonucleotide comprises one or moremodifications selected from: at least one modified sugar moiety, atleast one modified internucleoside linkage, at least one modifiednucleotide, and combinations thereof.
 11. The method of claim 10,wherein the one or more modifications comprise at least one modifiedsugar moiety selected from: a 2′-O-methoxyethyl modified sugar moiety, a2′-methoxy modified sugar moiety, a 2′-O-alkyl modified sugar moiety, abicyclic sugar moiety, and combinations thereof.
 12. The method of claim10, wherein the one or more modifications comprise at least one modifiedinternucleoside linkage selected from: a phosphorothioate,alkylphosphonate, phosphorodithioate, alkylphosphonothioate,phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate,carboxymethyl ester, and combinations thereof.
 13. The method of claim10, wherein the one or more modifications comprise at least one modifiednucleotide selected from: a peptide nucleic acid (PNA), a locked nucleicacid (LNA), an arabino-nucleic acid (FANA), an analogue, a derivative,and combinations thereof.
 14. The method of claim 2, wherein the atleast one oligonucleotide comprises at least one oligonucleotidesequences set forth as SEQ ID NOS: 10, 11, 16, 17, 19, 20, 21, 22, 23,24, 25 and
 27. 15. A method of upregulating a function of and/or theexpression of an Alpha-L-Iduronidase (IDUA) gene polynucleotide havingSEQ ID NO: 1 in mammalian cells or tissues in vivo or in vitrocomprising: contacting said cells or tissues with at least one shortinterfering RNA (siRNA) oligonucleotide 19 to 30 nucleotides in length,said at least one siRNA oligonucleotide being specific for a naturalantisense polynucleotide of an Alpha-L-Iduronidase (IDUA) polynucleotideselected from the group consisting of SEQ ID NOS: 2, 3 and 9; and,upregulating a function of and/or the expression of Alpha-L-Iduronidase(IDUA) in mammalian cells or tissues in vivo or in vitro.
 16. A methodof upregulating a function of and/or the expression ofAlpha-L-Iduronidase (IDUA) polynucleotide having SEQ ID NO: 1 inmammalian cells or tissues in vivo or in vitro comprising: contactingsaid cells or tissues with at least one single stranded antisenseoligonucleotide of about 12 to 30 nucleotides in length specific for anatural antisense strand of an Alpha-L-Iduronidase (IDUA) polynucleotidewherein said at least one antisense oligonucleotide has sequenceidentity to 12 to 30 consecutive nucleotides of at least one nucleicacid sequence set forth as SEQ ID NO: 1; and, upregulating the functionand/or expression of the Alpha-L-Iduronidase (IDUA) in mammalian cellsor tissues in vivo or in vitro.
 17. A method of increasing expression ofan IDUA polynucleotide or expression product thereof in a patient inneed of treatment thereof comprising administration of anoligonucleotide that is at least 50% identical to a reverse complementof a natural antisense transcript to said IDUA polynucleotide to saidpatient and wherein the increase in expression of IDUA relative to acontrol is greater than about ten (10) percent and said naturalantisense transcript is selected from SEQ ID NOS: 2, 3 and 9.